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1
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Li W, Yan ZF, Teng TS, Xiang XH. Mycobacterium tuberculosis Rv1043c regulates the inflammatory response by inhibiting the phosphorylation of TAK1. Int Microbiol 2024; 27:743-752. [PMID: 37676442 DOI: 10.1007/s10123-023-00428-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2023] [Revised: 08/24/2023] [Accepted: 08/29/2023] [Indexed: 09/08/2023]
Abstract
Mycobacterium tuberculosis can manipulate the host immunity through its effectors to ensure intracellular survival and colonization. Rv1043c has been identified as an effector potentially involved in M. tuberculosis pathogenicity. To explore the function of M. tuberculosis Rv1043c during infection, we overexpressed this protein in M. smegmatis, a non-pathogenic surrogate model in tuberculosis research. Here, we reported that Rv1043c enhanced mycobacterial survival and down-regulated the release of pro-inflammatory cytokines in macrophages and mice. In addition, Rv1043c inhibited the activation of MAPK and NF-κB signaling by preventing the phosphorylation of TAK1 indirectly. In conclusion, these data suggest that Rv1043c regulates the immune response and enhances the survival of recombinant M. smegmatis in vitro and in vivo.
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Affiliation(s)
- Wu Li
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, Sichuan, 641100, People's Republic of China
| | - Zi-Fei Yan
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, Sichuan, 641100, People's Republic of China
| | - Tie-Shan Teng
- School of Medical Sciences, College of Medicine, Henan University, Kaifeng, Henan, 475004, People's Republic of China
| | - Xiao-Hong Xiang
- School of Pharmacy, Chongqing Medical and Pharmaceutical College, Chongqing, 401331, People's Republic of China.
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2
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Flores-Valdez MA, Velázquez-Fernández JB, Pedroza-Roldán C, Aceves-Sánchez MDJ, Gutiérrez-Ortega A, López-Romero W, Barba-León J, Rodríguez-Campos J. Proteome and immunogenicity differences in BCG Pasteur ATCC 35734 and its derivative, the vaccine candidate BCGΔBCG1419c during planktonic growth in 7H9 and Proskauer Beck media. Tuberculosis (Edinb) 2024; 144:102432. [PMID: 38041962 DOI: 10.1016/j.tube.2023.102432] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Revised: 11/08/2023] [Accepted: 11/20/2023] [Indexed: 12/04/2023]
Abstract
Bacillus Calmette-Guèrin (BCG) remains as the only vaccine employed to prevent tuberculosis (TB) during childhood. Among factors likely contributing to the variable efficacy of BCG is the modification in its antigenic repertoire that may arise from in vitro growth conditions. Our vaccine candidate, BCGΔBCG1419c, improved protection against TB in mice and guinea pigs with bacteria grown in either 7H9 OADC Tween 80 or in Proskauer Beck Tween 80 media in independent studies. Here, we compared the proteomes of planktonic cultures of BCG and BCGΔBCG1419c, grown in both media. Further to this, we compared systemic immunogenicity ex vivo elicited by both types of BCG strains and cultures when used to vaccinate BALB/c mice. Both the parental strain BCG Pasteur ATCC 35734, and its isogenic mutant BCGΔBCG1419c, had several medium-dependent changes. Moreover, ex vivo immune responses to a multiantigenic (PPD) or a single antigenic (Ag85A) stimulus were also medium-dependent. Then, not only the presence or absence of the BCG1419c gene in our strains under study affected the proteome produced in vitro but also that this was affected by culture medium, potentially leading to changes in the capacity to induce ex vivo immune responses.
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Affiliation(s)
- Mario Alberto Flores-Valdez
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Av. Normalistas 800, Col. Colinas de la Normal, Guadalajara, Jalisco, 44270, Mexico.
| | | | - César Pedroza-Roldán
- Departamento de Medicina Veterinaria, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Zapopan, Mexico.
| | - Michel de Jesús Aceves-Sánchez
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Av. Normalistas 800, Col. Colinas de la Normal, Guadalajara, Jalisco, 44270, Mexico.
| | - Abel Gutiérrez-Ortega
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Av. Normalistas 800, Col. Colinas de la Normal, Guadalajara, Jalisco, 44270, Mexico.
| | - Wendy López-Romero
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Av. Normalistas 800, Col. Colinas de la Normal, Guadalajara, Jalisco, 44270, Mexico.
| | - Jeannette Barba-León
- Departamento de Salud Pública, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Zapopan, Jalisco, 45200, Mexico.
| | - Jacobo Rodríguez-Campos
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ), A. C., Unidad de Servicios Analíticos y Metrológicos, Av. Normalistas 800, Col. Colinas de la Normal, 44270, Guadalajara, Jalisco, Mexico.
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3
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Belardinelli JM, Arora D, Avanzi C, Wheat WH, Bryant JM, Spencer JS, Blundell TL, Parkhill J, Floto RA, Jackson M. Clinically relevant mutations in the PhoR sensor kinase of host-adapted Mycobacterium abscessus isolates impact response to acidic pH and virulence. Microbiol Spectr 2023; 11:e0158823. [PMID: 37874174 PMCID: PMC10715180 DOI: 10.1128/spectrum.01588-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 09/14/2023] [Indexed: 10/25/2023] Open
Abstract
IMPORTANCE Difficult-to-treat pulmonary infections caused by nontuberculous mycobacteria of the Mycobacterium abscessus group have been steadily increasing in the USA and globally. Owing to the relatively recent recognition of M. abscessus as a human pathogen, basic and translational research to address critical gaps in diagnosis, treatment, and prevention of diseases caused by this microorganism has been lagging behind that of the better-known mycobacterial pathogen, Mycobacterium tuberculosis. To begin unraveling the molecular mechanisms of pathogenicity of M. abscessus, we here focus on the study of a two-component regulator known as PhoPR which we found to be under strong evolutionary pressure during human lung infection. We show that PhoPR is activated at acidic pH and serves to regulate a defined set of genes involved in host adaptation. Accordingly, clinical isolates from chronically infected human lungs tend to hyperactivate this regulator enabling M. abscessus to escape macrophage killing.
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Affiliation(s)
- Juan M. Belardinelli
- Department of Microbiology, Immunology and Pathology, Mycobacteria Research Laboratories, Colorado State University, Fort Collins, Colorado, USA
| | - Divya Arora
- Department of Medicine, Molecular Immunity Unit, University of Cambridge, MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom
| | - Charlotte Avanzi
- Department of Microbiology, Immunology and Pathology, Mycobacteria Research Laboratories, Colorado State University, Fort Collins, Colorado, USA
| | - William H. Wheat
- Department of Microbiology, Immunology and Pathology, Mycobacteria Research Laboratories, Colorado State University, Fort Collins, Colorado, USA
| | - Josephine M. Bryant
- Department of Medicine, Molecular Immunity Unit, University of Cambridge, MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom
- University of Cambridge Centre for AI in Medicine, Cambridge, United Kingdom
| | - John S. Spencer
- Department of Microbiology, Immunology and Pathology, Mycobacteria Research Laboratories, Colorado State University, Fort Collins, Colorado, USA
| | - Tom L. Blundell
- Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - Julian Parkhill
- Wellcome Sanger Institute, Hinxton, United Kingdom
- Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
| | - R. Andres Floto
- Department of Medicine, Molecular Immunity Unit, University of Cambridge, MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom
- University of Cambridge Centre for AI in Medicine, Cambridge, United Kingdom
- Cambridge Centre for Lung Infection, Papworth Hospital, Cambridge, United Kingdom
| | - Mary Jackson
- Department of Microbiology, Immunology and Pathology, Mycobacteria Research Laboratories, Colorado State University, Fort Collins, Colorado, USA
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4
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Khan S, Ahmad F, Ansari MI, Ashfaque M, Islam MH, Khubaib M. Toxin-Antitoxin system of Mycobacterium tuberculosis: Roles beyond stress sensor and growth regulator. Tuberculosis (Edinb) 2023; 143:102395. [PMID: 37722233 DOI: 10.1016/j.tube.2023.102395] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2023] [Revised: 07/15/2023] [Accepted: 08/10/2023] [Indexed: 09/20/2023]
Abstract
The advent of effective drug regimen and BCG vaccine has significantly decreased the rate of morbidity and mortality of TB. However, lengthy treatment and slower recovery rate, as well as reactivation of the disease with the emergence of multi-drug, extensively-drug, and totally-drug resistance strains, pose a serious concern. The complexities associated are due to the highly evolved and complex nature of the bacterium itself. One of the unique features of Mycobacterium tuberculosis [M.tb] is that it has undergone reductive evolution while maintaining and amplified a few gene families. One of the critical gene family involved in the virulence and pathogenesis is the Toxin-Antitoxin system. These families are believed to harbor virulence signature and are strongly associated with various stress adaptations and pathogenesis. The M.tb TA systems are linked with growth regulation machinery during various environmental stresses. The genes of TA systems are differentially expressed in the host during an active infection, oxidative stress, low pH stress, and starvation, which essentially indicate their role beyond growth regulators. Here in this review, we have discussed different roles of TA gene families in various stresses and their prospective role at the host-pathogen interface, which could be exploited to understand the M.tb associated pathomechanisms better and further designing the new strategies against the pathogen.
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Affiliation(s)
- Saima Khan
- Department of Biosciences, Integral University, Lucknow, India
| | - Firoz Ahmad
- Department of Biosciences, Integral University, Lucknow, India
| | | | | | | | - Mohd Khubaib
- Department of Biosciences, Integral University, Lucknow, India.
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5
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Li LS, Yang L, Zhuang L, Ye ZY, Zhao WG, Gong WP. From immunology to artificial intelligence: revolutionizing latent tuberculosis infection diagnosis with machine learning. Mil Med Res 2023; 10:58. [PMID: 38017571 PMCID: PMC10685516 DOI: 10.1186/s40779-023-00490-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Accepted: 11/06/2023] [Indexed: 11/30/2023] Open
Abstract
Latent tuberculosis infection (LTBI) has become a major source of active tuberculosis (ATB). Although the tuberculin skin test and interferon-gamma release assay can be used to diagnose LTBI, these methods can only differentiate infected individuals from healthy ones but cannot discriminate between LTBI and ATB. Thus, the diagnosis of LTBI faces many challenges, such as the lack of effective biomarkers from Mycobacterium tuberculosis (MTB) for distinguishing LTBI, the low diagnostic efficacy of biomarkers derived from the human host, and the absence of a gold standard to differentiate between LTBI and ATB. Sputum culture, as the gold standard for diagnosing tuberculosis, is time-consuming and cannot distinguish between ATB and LTBI. In this article, we review the pathogenesis of MTB and the immune mechanisms of the host in LTBI, including the innate and adaptive immune responses, multiple immune evasion mechanisms of MTB, and epigenetic regulation. Based on this knowledge, we summarize the current status and challenges in diagnosing LTBI and present the application of machine learning (ML) in LTBI diagnosis, as well as the advantages and limitations of ML in this context. Finally, we discuss the future development directions of ML applied to LTBI diagnosis.
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Affiliation(s)
- Lin-Sheng Li
- Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Senior Department of Tuberculosis, the Eighth Medical Center of PLA General Hospital, Beijing, 100091, China
- Hebei North University, Zhangjiakou, 075000, Hebei, China
- Senior Department of Respiratory and Critical Care Medicine, the Eighth Medical Center of PLA General Hospital, Beijing, 100091, China
| | - Ling Yang
- Hebei North University, Zhangjiakou, 075000, Hebei, China
| | - Li Zhuang
- Hebei North University, Zhangjiakou, 075000, Hebei, China
| | - Zhao-Yang Ye
- Hebei North University, Zhangjiakou, 075000, Hebei, China
| | - Wei-Guo Zhao
- Senior Department of Respiratory and Critical Care Medicine, the Eighth Medical Center of PLA General Hospital, Beijing, 100091, China.
| | - Wen-Ping Gong
- Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Senior Department of Tuberculosis, the Eighth Medical Center of PLA General Hospital, Beijing, 100091, China.
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6
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Al-Jourani O, Benedict ST, Ross J, Layton AJ, van der Peet P, Marando VM, Bailey NP, Heunis T, Manion J, Mensitieri F, Franklin A, Abellon-Ruiz J, Oram SL, Parsons L, Cartmell A, Wright GSA, Baslé A, Trost M, Henrissat B, Munoz-Munoz J, Hirt RP, Kiessling LL, Lovering AL, Williams SJ, Lowe EC, Moynihan PJ. Identification of D-arabinan-degrading enzymes in mycobacteria. Nat Commun 2023; 14:2233. [PMID: 37076525 PMCID: PMC10115798 DOI: 10.1038/s41467-023-37839-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Accepted: 03/31/2023] [Indexed: 04/21/2023] Open
Abstract
Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.
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Affiliation(s)
- Omar Al-Jourani
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Samuel T Benedict
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Jennifer Ross
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Abigail J Layton
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Phillip van der Peet
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Victoria M Marando
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
- The Broad Institute of Harvard and MIT, Cambridge, MA, USA The Koch Integrative Cancer Research Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Nicholas P Bailey
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Tiaan Heunis
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Joseph Manion
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Francesca Mensitieri
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Aaron Franklin
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Javier Abellon-Ruiz
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Sophia L Oram
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Lauren Parsons
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Alan Cartmell
- Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
| | | | - Arnaud Baslé
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Matthias Trost
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Bernard Henrissat
- Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
- Department of Biotechnology and Biomedicine (DTU Bioengineering), Technical University of Denmark, 2800 Kgs, Lyngby, Denmark
| | - Jose Munoz-Munoz
- Microbial Enzymology Group, Department of Applied Sciences, Northumbria University, Newcastle upon Tyne, UK
| | - Robert P Hirt
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Laura L Kiessling
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Andrew L Lovering
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Spencer J Williams
- School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Elisabeth C Lowe
- Newcastle University Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
| | - Patrick J Moynihan
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
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7
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D'Souza C, Kishore U, Tsolaki AG. The PE-PPE Family of Mycobacterium tuberculosis: Proteins in Disguise. Immunobiology 2023; 228:152321. [PMID: 36805109 DOI: 10.1016/j.imbio.2022.152321] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Revised: 12/22/2022] [Accepted: 12/27/2022] [Indexed: 12/31/2022]
Abstract
Mycobacterium tuberculosis has thrived in parallel with humans for millennia, and despite our efforts, M. tuberculosis continues to plague us, currently infecting a third of the world's population. The success of M. tuberculosis has recently been attributed, in part, to the PE-PPE family; a unique collection of 168 proteins fundamentally involved in the pathogenesis of M. tuberculosis. The PE-PPE family proteins have been at the forefront of intense research efforts since their discovery in 1998 and whilst our knowledge and understanding has significantly advanced over the last two decades, many important questions remain to be elucidated. This review consolidates and examines the vast body of existing literature regarding the PE-PPE family proteins, with respect to the latest developments in elucidating their evolution, structure, subcellular localisation, function, and immunogenicity. This review also highlights significant inconsistencies and contradictions within the field. Additionally, possible explanations for these knowledge gaps are explored. Lastly, this review poses many important questions, which need to be addressed to complete our understanding of the PE-PPE family, as well as highlighting the challenges associated with studying this enigmatic family of proteins. Further research into the PE-PPE family, together with technological advancements in genomics and proteomics, will undoubtedly improve our understanding of the pathogenesis of M. tuberculosis, as well as identify key targets/candidates for the development of novel drugs, diagnostics, and vaccines.
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Affiliation(s)
- Christopher D'Souza
- Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London, Uxbridge UB8 3PH, United Kingdom
| | - Uday Kishore
- Department of Veterinary Medicine, United Arab Emirates University, Al Ain, United Arab Emirates
| | - Anthony G Tsolaki
- Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London, Uxbridge UB8 3PH, United Kingdom.
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8
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Matsumura K, Takaki S, Kirikae T. Mycobacterial protein PE_PGRS30 induces macrophage apoptosis through prohibitin 2 mitochondrial function interference. Front Microbiol 2023; 14:1080369. [PMID: 36778852 PMCID: PMC9911437 DOI: 10.3389/fmicb.2023.1080369] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 01/10/2023] [Indexed: 01/28/2023] Open
Abstract
PE_PGRS30 belongs to the PE_PGRS protein family and is characterized by a conserved Pro-Glu (PE) domain and a typically polymorphic GC-rich sequence (PGRS) domain. PE_PGRS30 is a virulence factor of Mycobacterium tuberculosis that induces macrophage cell death. We found that RAW264.7 cells and murine alveolar macrophages underwent apoptosis in response to PE_PGRS30. The host protein prohibitin 2 (PHB2) was identified as a target molecule. PE_PGRS30 and PHB2 interact via the PGRS domain and mitochondrial targeting sequence, respectively. PHB2 overexpression reduced macrophage apoptosis in response to PE_PGRS30. PE_PGRS30 co-localized with PHB2, not in mitochondria, but in lysosomes. The maintenance of mitochondrial structure by PHB2 was impaired in response to the PGRS domain. These results indicated that PE_PGRS30 reduces PHB2 in mitochondria, resulting in mitochondrial dysfunction and cellular apoptosis.
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Affiliation(s)
- Kazunori Matsumura
- Department of Immune Regulation, Research Institute, National Center for Global Health and Medicine, Chiba, Japan
| | - Satoshi Takaki
- Department of Immune Regulation, Research Institute, National Center for Global Health and Medicine, Chiba, Japan
| | - Teruo Kirikae
- Graduate School of Medicine, Juntendo University, Tokyo, Japan,*Correspondence: Teruo Kirikae,
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9
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Aylan B, Botella L, Gutierrez MG, Santucci P. High content quantitative imaging of Mycobacterium tuberculosis responses to acidic microenvironments within human macrophages. FEBS Open Bio 2022. [PMID: 36520007 DOI: 10.1002/2211-5463.13537] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2022] [Revised: 11/30/2022] [Accepted: 12/04/2022] [Indexed: 12/23/2022] Open
Abstract
Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) have evolved diverse strategies to counteract macrophage defence mechanisms including phagolysosomal biogenesis. Within macrophages, Mtb initially resides inside membrane-bound phagosomes that interact with lysosomes and become acidified. The ability of Mtb to control and subvert the fusion between phagosomes and lysosomes plays a key role in the pathogenesis of tuberculosis. Therefore, understanding how pathogens interact with the endolysosomal network and cope with intracellular acidification is important to better understand the disease. Here, we describe in detail the use of fluorescence microscopy-based approaches to investigate Mtb responses to acidic environments in cellulo. We report high-content imaging modalities to probe Mtb sensing of external pH or visualise in real-time Mtb intrabacterial pH within infected human macrophages. We discuss various methodologies with step-by-step analyses that enable robust image-based quantifications. Finally, we highlight the advantages and limitations of these different approaches and discuss potential alternatives that can be applied to further investigate Mtb-host cell interactions. These methods can be adapted to study host-pathogen interactions in different biological systems and experimental settings. Altogether, these approaches represent a valuable tool to further broaden our understanding of the cellular and molecular mechanisms underlying intracellular pathogen survival.
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Affiliation(s)
- Beren Aylan
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK
| | - Laure Botella
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK
| | - Maximiliano G Gutierrez
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK
| | - Pierre Santucci
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK
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10
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Moopanar K, Nyide ANG, Senzani S, Mvubu NE. Clinical strains of Mycobacterium tuberculosis exhibit differential lipid metabolism-associated transcriptome changes in in vitro cholesterol and infection models. Pathog Dis 2022; 81:6889515. [PMID: 36509392 PMCID: PMC9936260 DOI: 10.1093/femspd/ftac046] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2022] [Revised: 10/30/2022] [Accepted: 12/09/2022] [Indexed: 12/15/2022] Open
Abstract
Many studies have identified host-derived lipids, characterised by the abundance of cholesterol, as a major source of carbon nutrition for Mycobacterium tuberculosis during infection. Members of the Mycobacterium tuberculosis complex are biologically different with regards to degree of disease, host range, pathogenicity and transmission. Therefore, the current study aimed at elucidating transcriptome changes during early infection of pulmonary epithelial cells and on an in vitro cholesterol-rich minimal media, in M. tuberculosis clinical strains F15/LAM4/KZN and Beijing, and the laboratory H37Rv strain. Infection of pulmonary epithelial cells elicited the upregulation of fadD28 and hsaC in both the F15/LAM4/KZN and Beijing strains and the downregulation of several other lipid-associated genes. Growth curve analysis revealed F15/LAM4/KZN and Beijing to be slow growers in 7H9 medium and cholesterol-supplemented media. RNA-seq analysis revealed strain-specific transcriptomic changes, thereby affecting different metabolic processes in an in vitro cholesterol model. The differential expression of these genes suggests that the genetically diverse M. tuberculosis clinical strains exhibit strain-specific behaviour that may influence their ability to metabolise lipids, specifically cholesterol, which may account for phenotypic differences observed during infection.
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Affiliation(s)
- Kynesha Moopanar
- Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa
| | - Asanda Nomfundo Graduate Nyide
- Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa
| | - Sibusiso Senzani
- Medical Microbiology, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, 1st floor, Doris Duke Medical Research Institute, Congella, Private Bag 7, Durban, 4013, South Africa
| | - Nontobeko Eunice Mvubu
- Corresponding author. Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa.Tel: +27 31 260 7404; E-mail:
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11
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Xue S, Ma J, Li SS, Fan S, Cai Y, Li J, Fu X, Deng Z, Sun QH, Sun YC, Ma W. Mining of the Novel Virulent ATP-Binding Cassette Importers in Mycobacterium abscessus by Comparative Genomic Strategy. Microb Drug Resist 2022; 28:1057-1064. [DOI: 10.1089/mdr.2021.0450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Affiliation(s)
- Song Xue
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
| | - Jian Ma
- Department of Respiratory and Critical Care Medicine, Shanghai Pulmony Hospital, Shanghai, P.R. China
| | - Si-Shang Li
- MOH Key Laboratory of Systems Biology of Pathogens, Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Pathogen Biology, Beijing, P.R. China
| | - Shuxuan Fan
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
| | - YiChun Cai
- School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
| | - Jiahao Li
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
| | - Xiang Fu
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
| | - ZiXin Deng
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
| | - Qiu Hong Sun
- Department of Respiratory and Critical Care Medicine, Shanghai Pulmony Hospital, Shanghai, P.R. China
| | - Yi-Cheng Sun
- MOH Key Laboratory of Systems Biology of Pathogens, Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Pathogen Biology, Beijing, P.R. China
| | - Wei Ma
- State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, P.R. China
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12
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Santucci P, Aylan B, Botella L, Bernard EM, Bussi C, Pellegrino E, Athanasiadi N, Gutierrez MG. Visualizing Pyrazinamide Action by Live Single-Cell Imaging of Phagosome Acidification and Mycobacterium tuberculosis pH Homeostasis. mBio 2022; 13:e0011722. [PMID: 35323041 PMCID: PMC9040869 DOI: 10.1128/mbio.00117-22] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Accepted: 02/24/2022] [Indexed: 01/28/2023] Open
Abstract
Mycobacterium tuberculosis segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and M. tuberculosis intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that M. tuberculosis can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH), or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by disrupting M. tuberculosis intrabacterial pH homeostasis in cellulo. By using M. tuberculosis mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. IMPORTANCE We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug. Our observations can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies for tuberculosis that target heterogenous bacterial populations within the host.
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Affiliation(s)
- Pierre Santucci
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Beren Aylan
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Laure Botella
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Elliott M. Bernard
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Claudio Bussi
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Enrica Pellegrino
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Natalia Athanasiadi
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Maximiliano G. Gutierrez
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, United Kingdom
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13
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Sharma S, Sharma M. Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) proteins of Mycobacterium tuberculosis: The multifaceted immune-modulators. Acta Trop 2021; 222:106035. [PMID: 34224720 DOI: 10.1016/j.actatropica.2021.106035] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Revised: 06/23/2021] [Accepted: 06/29/2021] [Indexed: 12/30/2022]
Abstract
The PE/PPE proteins encoded by seven percent (7%) of Mycobacterium tuberculosis (Mtb) genome are the chief constituents to pathogen's virulence reservoir. The fact that these genes have evolved along ESX secretory system in pathogenic Mtb strains make their investigation very intriguing. There is lot of speculation about the prominent role of these proteins at host pathogen interface and in disease pathogenesis. Nevertheless, the exact function of PE/PPE proteins still remains a mystery which calls for further research targeting these proteins. This article is an effort to document all the facts known so far with regard to these unique proteins which involves their origin, evolution, transcriptional control, and most important their role as host immune-modulators. Our understanding strongly points towards the versatile nature of these PE/PPE proteins as Mtb's host immune sensors and as decisive factors in shaping the outcome of infection. Further investigation on these proteins will surely pave way for newer and effective vaccines and therapeutics to control Tuberculosis (TB).
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Affiliation(s)
- Sadhna Sharma
- DS Kothari Central Interdisciplinary Research Centre and Department of Zoology, Miranda House, University of Delhi, Delhi 110007, India.
| | - Monika Sharma
- DS Kothari Central Interdisciplinary Research Centre and Department of Zoology, Miranda House, University of Delhi, Delhi 110007, India.
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14
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Richardson W, Kang GW, Lee HJ, Kwon KM, Kim S, Kim HJ. Chasing the structural diversity of the transcription regulator Mycobacterium tuberculosis HigA2. IUCRJ 2021; 8:823-832. [PMID: 34584743 PMCID: PMC8420761 DOI: 10.1107/s2052252521007715] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Accepted: 07/28/2021] [Indexed: 06/13/2023]
Abstract
Transcription factors are the primary regulators of gene expression and recognize specific DNA sequences under diverse physiological conditions. Although they are vital for many important cellular processes, it remains unclear when and how transcription factors and DNA interact. The antitoxin from a toxin-antitoxin system is an example of negative transcriptional autoregulation: during expression of the cognate toxin it is suppressed through binding to a specific DNA sequence. In the present study, the antitoxin HigA2 from Mycobacterium tuberculosis M37Rv was structurally examined. The crystal structure of M. tuberculosis HigA2 comprises three sections: an N-terminal autocleavage region, an α-helix bundle which contains an HTH motif, and a C-terminal β-lid. The N-terminal region is responsible for toxin binding, but was shown to cleave spontaneously in its absence. The HTH motif performs a key role in DNA binding, with the C-terminal β-lid influencing the interaction by mediating the distance between the motifs. However, M. tuberculosis HigA2 exhibits a unique coordination of the HTH motif and no DNA-binding activity is detected. Three crystal structures of M. tuberculosis HigA2 show a flexible alignment of the HTH motif, which implies that the motif undergoes structural rearrangement to interact with DNA. This study reveals the molecular mechanisms of how transcription factors interact with partner proteins or DNA.
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Affiliation(s)
- William Richardson
- Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom
| | - Gyun Won Kang
- College of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
| | - Hee Joong Lee
- College of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
| | - Kang Mu Kwon
- College of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
| | - Saron Kim
- College of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
| | - Hyo Jung Kim
- Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom
- College of Pharmacy, Woosuk University, Wanju 55338, Republic of Korea
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15
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Asaad M, Kaisar Ali M, Abo-Kadoum MA, Lambert N, Gong Z, Wang H, Uae M, Nazou SAE, Kuang Z, Xie J. Mycobacterium tuberculosis PPE10 (Rv0442c) alters host cell apoptosis and cytokine profile via linear ubiquitin chain assembly complex HOIP-NF-κB signaling axis. Int Immunopharmacol 2021; 94:107363. [PMID: 33667868 DOI: 10.1016/j.intimp.2020.107363] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Revised: 12/27/2020] [Accepted: 12/30/2020] [Indexed: 12/20/2022]
Abstract
Tuberculosis caused by Mycobacterium tuberculosis infection remains one of the top ten causes of deaths worldwide. M. tuberculosis genome devoted 10% capacity for highly repeated PE/PPE genes family. To explore the role of PPE10 in host-pathogen interaction, PPE10 encoding gene Rv0442c was heterologously expressed in the nonpathogenic M. smegmatis strain. PPE10 altered the bacterial cell surface properties, colony morphology, and biofilm formation. Ms_PPE10 showed more resistance to stress conditions such as diamide, and low pH, as well as higher survival within the macrophage. Moreover, the host's cell apoptosis was regulated via decreased expression of caspases, IL-1, IL-6, and TNF-α through the Linear Ubiquitin Chain Assembly Complex (LUBAC) HOIP-NF-κB signaling axis. The study revealed novel insights into the mechanism of action of the PPE family.
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Affiliation(s)
- Mohammed Asaad
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China; Department of Biotechnology, Faculty of Science and Technology, Omdurman Islamic University, Omdurman, Khartoum, Sudan
| | - Md Kaisar Ali
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - M A Abo-Kadoum
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China; Department of Botany and Microbiology, Faculty of Science, Al-Azhar University Assuit branch, Egypt
| | - Nzungize Lambert
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - Zhen Gong
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - Hao Wang
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - Moure Uae
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - Stech A E Nazou
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - Zhongmei Kuang
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China
| | - Jianping Xie
- State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Institute of Modern Biopharmaceuticals, Southwest University, Chongqing, PR China.
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16
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Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis. Int J Mol Sci 2021; 22:ijms22041560. [PMID: 33557133 PMCID: PMC7913854 DOI: 10.3390/ijms22041560] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2020] [Revised: 01/21/2021] [Accepted: 01/29/2021] [Indexed: 12/26/2022] Open
Abstract
The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.
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17
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Chang DPS, Guan XL. Metabolic Versatility of Mycobacterium tuberculosis during Infection and Dormancy. Metabolites 2021; 11:88. [PMID: 33540752 PMCID: PMC7913082 DOI: 10.3390/metabo11020088] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2020] [Revised: 01/16/2021] [Accepted: 01/29/2021] [Indexed: 12/18/2022] Open
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful intracellular pathogen with the ability to withstand harsh conditions and reside long-term within its host. In the dormant and persistent states, the bacterium tunes its metabolism and is able to resist the actions of antibiotics. One of the main strategies Mtb adopts is through its metabolic versatility-it is able to cometabolize a variety of essential nutrients and direct these nutrients simultaneously to multiple metabolic pathways to facilitate the infection of the host. Mtb further undergo extensive remodeling of its metabolic pathways in response to stress and dormancy. In recent years, advancement in systems biology and its applications have contributed substantially to a more coherent view on the intricate metabolic networks of Mtb. With a more refined appreciation of the roles of metabolism in mycobacterial infection and drug resistance, and the success of drugs targeting metabolism, there is growing interest in further development of anti-TB therapies that target metabolism, including lipid metabolism and oxidative phosphorylation. Here, we will review current knowledge revolving around the versatility of Mtb in remodeling its metabolism during infection and dormancy, with a focus on central carbon metabolism and lipid metabolism.
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Affiliation(s)
| | - Xue Li Guan
- Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921, Singapore;
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18
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The ATP-Binding Cassette (ABC) Transport Systems in Mycobacterium tuberculosis: Structure, Function, and Possible Targets for Therapeutics. BIOLOGY 2020; 9:biology9120443. [PMID: 33291531 PMCID: PMC7761784 DOI: 10.3390/biology9120443] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/10/2020] [Revised: 11/06/2020] [Accepted: 11/12/2020] [Indexed: 12/22/2022]
Abstract
Simple Summary Mycobacterium tuberculosis is a bacterium of great medical importance because it causes tuberculosis, a disease that affects millions of people worldwide. Two important features are related to this bacterium: its ability to infect and survive inside the host, minimizing the immune response, and the burden of clinical isolates that are highly resistant to antibiotics treatment. These two phenomena are directly affected by cell envelope proteins, such as proteins from the ATP-Binding Cassette (ABC transporters) superfamily. In this review, we have compiled information on all the M. tuberculosis ABC transporters described so far, both from a functional and structural point of view, and show their relevance for the bacillus and the potential targets for studies aiming to control the microorganism and structural features. Abstract Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), a disease that affects millions of people in the world and that is associated with several human diseases. The bacillus is highly adapted to infect and survive inside the host, mainly because of its cellular envelope plasticity, which can be modulated to adapt to an unfriendly host environment; to manipulate the host immune response; and to resist therapeutic treatment, increasing in this way the drug resistance of TB. The superfamily of ATP-Binding Cassette (ABC) transporters are integral membrane proteins that include both importers and exporters. Both types share a similar structural organization, yet only importers have a periplasmic substrate-binding domain, which is essential for substrate uptake and transport. ABC transporter-type importers play an important role in the bacillus physiology through the transport of several substrates that will interfere with nutrition, pathogenesis, and virulence. Equally relevant, exporters have been involved in cell detoxification, nutrient recycling, and antibiotics and drug efflux, largely affecting the survival and development of multiple drug-resistant strains. Here, we review known ABC transporters from M. tuberculosis, with particular focus on the diversity of their structural features and relevance in infection and drug resistance.
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19
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Madhvi A, Mishra H, Chegou NN, Tromp G, Van Heerden CJ, Pietersen RD, Leisching G, Baker B. Distinct host-immune response toward species related intracellular mycobacterial killing: A transcriptomic study. Virulence 2020; 11:170-182. [PMID: 32052695 PMCID: PMC7051142 DOI: 10.1080/21505594.2020.1726561] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2019] [Revised: 01/01/2020] [Accepted: 01/03/2020] [Indexed: 01/10/2023] Open
Abstract
The comparison of the host immune response when challenged with pathogenic and nonpathogenic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.
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Affiliation(s)
- Abhilasha Madhvi
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Hridesh Mishra
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Novel N. Chegou
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Gerard Tromp
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
- South African Tuberculosis Bioinformatics Initiative (SATBBI), Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
- Centre for Bioinformatics and Computational Biology, Stellenbosch University, Cape Town, South Africa
| | - Carel J. Van Heerden
- DNA Sequencing Unit, Central Analytical Facility (CAF), Stellenbosch University, Stellenbosch, South Africa
| | - R. D. Pietersen
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Gina Leisching
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Bienyameen Baker
- NRF-DST Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council Centre for Tuberculosis Research; Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
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20
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Dolasia K, Nazar F, Mukhopadhyay S. Mycobacterium tuberculosis PPE18 protein inhibits MHC class II antigen presentation and B cell response in mice. Eur J Immunol 2020; 51:603-619. [PMID: 33084017 DOI: 10.1002/eji.201848071] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Revised: 09/22/2020] [Accepted: 10/19/2020] [Indexed: 01/18/2023]
Abstract
PPE18 protein belongs to PE/PPE family of Mycobacterium tuberculosis. We reported earlier that PPE18 protein provides survival advantage to M. tuberculosis during infection. In the current study, we found that PPE18 inhibits MHC class II-mediated antigen presentation by macrophages in a dose-dependent manner without affecting the surface level of MHC class II or co-stimulatory molecules. PPE18 does not affect antigen uptake or presentation of preprocessed peptide by macrophages. Antigen degradation was found to be inhibited by PPE18 protein due to perturbation in phagolysosomal acidification. PPE18-mediated inhibition of MHC class II antigen presentation caused poorer activation of CD4 T cells. Mice infected with M. smegmatis expressing PPE18 exhibited reduced maturation and activation of B cells and had decreased Mycobacteria-specific antibody titers. Thus M. tuberculosis probably utilizes PPE18 to inhibit MHC class II antigen presentation causing poorer activation of adaptive immune responses. This study may be useful in understanding host-pathogen interaction and open up directions of designing novel therapeutics targeting PPE18 to tackle this nefarious pathogen.
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Affiliation(s)
- Komal Dolasia
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India.,Graduate Studies, Manipal Academy of Higher Education, Manipal, India
| | - Faiza Nazar
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India
| | - Sangita Mukhopadhyay
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India
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21
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Heterologous Production of 1-Tuberculosinyladenosine in Mycobacterium kansasii Models Pathoevolution towards the Transcellular Lifestyle of Mycobacterium tuberculosis. mBio 2020; 11:mBio.02645-20. [PMID: 33082253 PMCID: PMC7587436 DOI: 10.1128/mbio.02645-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2-/- mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation.IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.
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22
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Sharma T, Grover S, Arora N, P M, Ehtesham NZ, Hasnain SE. PGRS Domain of Rv0297 of Mycobacterium tuberculosis Is Involved in Modulation of Macrophage Functions to Favor Bacterial Persistence. Front Cell Infect Microbiol 2020; 10:451. [PMID: 33042856 PMCID: PMC7517703 DOI: 10.3389/fcimb.2020.00451] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2020] [Accepted: 07/23/2020] [Indexed: 01/04/2023] Open
Abstract
Mycobacterium tuberculosis (M. tb) Rv0297-encoded PE_PGRS5 has been known to be expressed at the later stages of infection and in acidified phagosomes during transcriptome and proteomic studies. The possible role of Rv0297 in the modulation of phagosomal maturation and in providing protection against a microbicidal environment has been hypothesized. We show that Rv0297PGRS is involved in modulating the calcium homeostasis of macrophages followed by impedance of the phagolysosomal acidification process. This is evident from the downregulation of the late endosomal markers (Rab7 and cathepsin D) in the macrophages infected with recombinant Mycobacterium smegmatis (rM.smeg)—M.smeg_Rv0297 and M.smeg_Rv0297PGRS—or treated with recombinant Rv0297PGRS protein. Macrophages infected with rM.smeg expressing Rv0297 produce nitric oxide and undergo apoptosis, which may aid in the dissemination of pathogen in the later stages of infection. Rv0297 was also found to be involved in rescuing the bacterium from oxidative and hypoxic stress employed by macrophages and augmented the survivability of the recombinant bacterium. These results attribute to the functional significance of this protein in M.tb virulence mechanism. The fact that this protein gets expressed at the later stages of lung granulomas during M.tb infection suggests that the bacterium possibly employs Rv0297 as its dissemination and survival strategy.
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Affiliation(s)
- Tarina Sharma
- Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India
| | - Sonam Grover
- Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Naresh Arora
- Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Manjunath P
- ICMR-National Institute of Pathology, New Delhi, India
| | | | - Seyed Ehtesham Hasnain
- Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India.,Dr. Reddy's Institute of Life Sciences, Hyderabad, India
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23
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Yang Y, Xu P, He P, Shi F, Tang Y, Guan C, Zeng H, Zhou Y, Song Q, Zhou B, Jiang S, Shao C, Sun J, Yang Y, Wang X, Song H. Mycobacterial PPE13 activates inflammasome by interacting with the NATCH and LRR domains of NLRP3. FASEB J 2020; 34:12820-12833. [PMID: 32738179 DOI: 10.1096/fj.202000200rr] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2020] [Revised: 06/09/2020] [Accepted: 07/16/2020] [Indexed: 12/24/2022]
Abstract
Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marinum, can trigger NLRP3 inflammasome activation leading to maturation and secretion of interleukin 1β (IL-1β). However, the mycobacterial factors involved in the activation of NLRP3 inflammasome are not fully understood. Here, we identified that the PPE family protein PPE13 was responsible for the induction of IL-1β secretion in a NLRP3 inflammasome-dependent manner. We found that the recombinant Mycobacterium smegmatis expressing PPE13 activates NLRP3 inflammasome, thereby inducing caspase-1 cleavage and IL-1β secretion in J774A.1, BMDMs, and THP-1 macrophages. To examine whether this inflammasome activation was triggered by PPE13 rather than components of M. smegmatis, PPE13 was introduced into the aforementioned macrophages by lentivirus as a delivery vector. Similarly, this led to the activation of NLRP3 inflammasome, indicating that PPE13 is a direct activator of NLRP3 cascade. We further demonstrated that the NLRP3 complex activated the inflammasome cascade, and the assembly of this complex was facilitated by PPE13 through interacting with the LRR and NATCH domains of NLRP3. Finally, we found that all PPE13 proteins isolated from M. tuberculosis, M. bovis, and M. marinum can activate NLRP3 inflammasome through binding to NLRP3, which requires C-terminal repetitive MPTR domain of PPE13. Thus, we, for the first time, revealed that PPE13 triggers the inflammasome-response by interacting with the MPTR domain of PPE13 and the LRR and NATCH domains of NLRP3. These findings provide a novel perspective on the function of PPE proteins in the immune system during mycobacteria invasion.
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Affiliation(s)
- Yang Yang
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Pianpian Xu
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Ping He
- National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Fushan Shi
- Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, China
| | - Yiran Tang
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Chiyu Guan
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Huan Zeng
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Yingshan Zhou
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Quanjiang Song
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Bin Zhou
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Sheng Jiang
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Chunyan Shao
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Jing Sun
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Yongchun Yang
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Xiaodu Wang
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
| | - Houhui Song
- College of Animal Science and Technology, College of Veterinary Medicine, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, Zhejiang A&F University, Hangzhou, China
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24
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la Cruz MAD, Ares MA, Rodríguez-Valverde D, Vallejo-Cardona AA, Flores-Valdez MA, Núñez IDC, Aceves-Sánchez MDJ, Lira-Chávez J, Rodríguez-Campos J, Bravo-Madrigal J. Transcriptional and Mycolic Acid Profiling in Mycobacterium bovis BCG In Vitro Show an Effect for c-di-GMP and Overlap between Dormancy and Biofilms. J Microbiol Biotechnol 2020; 30:811-821. [PMID: 32238759 PMCID: PMC9728378 DOI: 10.4014/jmb.1911.11043] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Accepted: 03/12/2020] [Indexed: 12/15/2022]
Abstract
Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.
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Affiliation(s)
- Miguel A. De la Cruz
- Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional (CMN) Siglo XXI, Instituto Mexicano de Seguro Social (IMSS), Ciudad de México, México
| | - Miguel A. Ares
- Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional (CMN) Siglo XXI, Instituto Mexicano de Seguro Social (IMSS), Ciudad de México, México
| | - Diana Rodríguez-Valverde
- Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional (CMN) Siglo XXI, Instituto Mexicano de Seguro Social (IMSS), Ciudad de México, México
| | - Alba Adriana Vallejo-Cardona
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C., Biotecnología Médica y Farmacéutica, Av. Normalistas No. 800. Colinas de la Normal, C.P. 4470 Guadalajara, Jalisco, México,Alba Adriana Vallejo-Cardona Phone: +52-33-33-45-52-00 E-mail:
| | - Mario Alberto Flores-Valdez
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C., Biotecnología Médica y Farmacéutica, Av. Normalistas No. 800. Colinas de la Normal, C.P. 4470 Guadalajara, Jalisco, México,Corresponding authors Mario Alberto Flores-Valdez Phone: +52-33-33-45-52-00 E-mail:
| | - Iris Denisse Cota Núñez
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C., Biotecnología Médica y Farmacéutica, Av. Normalistas No. 800. Colinas de la Normal, C.P. 4470 Guadalajara, Jalisco, México
| | - Michel de Jesús Aceves-Sánchez
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C., Biotecnología Médica y Farmacéutica, Av. Normalistas No. 800. Colinas de la Normal, C.P. 4470 Guadalajara, Jalisco, México
| | - Jonahtan Lira-Chávez
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C., Biotecnología Médica y Farmacéutica, Av. Normalistas No. 800. Colinas de la Normal, C.P. 4470 Guadalajara, Jalisco, México
| | - Jacobo Rodríguez-Campos
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C, Unidad de Servicios Analíticos y Metrológicos, Av. Normalistas No. 800. Colinas de la Normal, C.P. 44270 Guadalajara, Jalisco, México
| | - Jorge Bravo-Madrigal
- Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco (CIATEJ) A.C., Biotecnología Médica y Farmacéutica, Av. Normalistas No. 800. Colinas de la Normal, C.P. 4470 Guadalajara, Jalisco, México
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25
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Sapriel G, Brosch R. Shared Pathogenomic Patterns Characterize a New Phylotype, Revealing Transition toward Host-Adaptation Long before Speciation of Mycobacterium tuberculosis. Genome Biol Evol 2020; 11:2420-2438. [PMID: 31368488 PMCID: PMC6736058 DOI: 10.1093/gbe/evz162] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/09/2019] [Indexed: 02/06/2023] Open
Abstract
Tuberculosis remains one of the deadliest infectious diseases of humanity. To better understand the evolutionary history of host-adaptation of tubercle bacilli (MTB), we sought for mycobacterial species that were more closely related to MTB than the previously used comparator species Mycobacterium marinum and Mycobacterium kansasii. Our phylogenomic approach revealed some recently sequenced opportunistic mycobacterial pathogens, Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense, to constitute a common clade with MTB, hereafter called MTB-associated phylotype (MTBAP), from which MTB have emerged. Multivariate and clustering analyses of genomic functional content revealed that the MTBAP lineage forms a clearly distinct cluster of species that share common genomic characteristics, such as loss of core genes, shift in dN/dS ratios, and massive expansion of toxin–antitoxin systems. Consistently, analysis of predicted horizontal gene transfer regions suggests that putative functions acquired by MTBAP members were markedly associated with changes in microbial ecology, for example adaption to intracellular stress resistance. Our study thus considerably deepens our view on MTB evolutionary history, unveiling a decisive shift that promoted conversion to host-adaptation among ancestral founders of the MTBAP lineage long before Mycobacterium tuberculosis has adapted to the human host.
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Affiliation(s)
- Guillaume Sapriel
- UFR des Sciences de La Santé, Université de Versailles St. Quentin, Montigny le Bretonneux, France.,Atelier de Bioinformatique, ISYEB, UMR 7205, Paris, France
| | - Roland Brosch
- Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, CNRS UMR 3525, Paris, France
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26
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Stårsta M, Hammarlöf DL, Wäneskog M, Schlegel S, Xu F, Heden Gynnå A, Borg M, Herschend S, Koskiniemi S. RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium. PLoS Genet 2020; 16:e1008607. [PMID: 32053596 PMCID: PMC7043789 DOI: 10.1371/journal.pgen.1008607] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2019] [Revised: 02/26/2020] [Accepted: 01/12/2020] [Indexed: 11/19/2022] Open
Abstract
RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages. Bacteria that reside and multiply inside of phagocytic cells are hard to treat with common antibiotics, partly because subpopulations of bacteria are non-growing. Very little is known about how bacteria regulate their growth in the phagocytic vesicle. We show that RHS elements, previously known to function as mobilizable toxins that inhibit growth of neighboring bacteria, also function as internally expressed toxin-antitoxin systems that regulate Salmonella Typhimurium growth in macrophages. RHS elements were discovered more than 30 years ago, but their role in biology has long remained unclear even though they are some of the most positively selected genes known. Our results suggest an explanation to why rhs genes are under such strong positive selection in addition to suggesting a novel function for these toxins in regulating bacterial growth.
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Affiliation(s)
- Magnus Stårsta
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Disa L. Hammarlöf
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Marcus Wäneskog
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Susan Schlegel
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Feifei Xu
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Arvid Heden Gynnå
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Malin Borg
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Sten Herschend
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
| | - Sanna Koskiniemi
- Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
- * E-mail:
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27
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Soni DK, Dubey SK, Bhatnagar R. ATP-binding cassette (ABC) import systems of Mycobacterium tuberculosis: target for drug and vaccine development. Emerg Microbes Infect 2020; 9:207-220. [PMID: 31985348 PMCID: PMC7034087 DOI: 10.1080/22221751.2020.1714488] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Nutrient procurement specifically from nutrient-limiting environment is essential for pathogenic bacteria to survive and/or persist within the host. Long-term survival or persistent infection is one of the main reasons for the overuse of antibiotics, and contributes to the development and spread of antibiotic resistance. Mycobacterium tuberculosis is known for long-term survival within the host, and develops multidrug resistance. Before and during infection, the pathogen encounters various harsh environmental conditions. To cope up with such nutrient-limiting conditions, it is crucial to uptake essential nutrients such as ions, sugars, amino acids, peptides, and metals, necessary for numerous vital biological activities. Among the various types of transporters, ATP-binding cassette (ABC) importers are essentially unique to bacteria, accessible as drug targets without penetrating the cytoplasmic membrane, and offer an ATP-dependent gateway into the cell by mimicking substrates of the importer and designing inhibitors against substrate-binding proteins, ABC importers endeavour for the development of successful drug candidates and antibiotics. Alternatively, the production of antibodies against substrate-binding proteins could lead to vaccine development. In this review, we will emphasize the role of M. tuberculosis ABC importers for survival and virulence within the host. Furthermore, we will elucidate their unique characteristics to discover emerging therapies to combat tuberculosis.
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Affiliation(s)
- Dharmendra Kumar Soni
- Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
| | - Suresh Kumar Dubey
- Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, India
| | - Rakesh Bhatnagar
- Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
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28
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Butler RE, Smith AA, Mendum TA, Chandran A, Wu H, Lefrançois L, Chambers M, Soldati T, Stewart GR. Mycobacterium bovis uses the ESX-1 Type VII secretion system to escape predation by the soil-dwelling amoeba Dictyostelium discoideum. ISME JOURNAL 2020; 14:919-930. [PMID: 31896783 PMCID: PMC7082363 DOI: 10.1038/s41396-019-0572-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/11/2019] [Revised: 11/26/2019] [Accepted: 12/11/2019] [Indexed: 12/11/2022]
Abstract
Mycobacterium bovis is the causative agent of bovine tuberculosis and the predominant cause of zoonotic tuberculosis in people. Bovine tuberculosis occurs in farmed cattle but also in a variety of wild animals, which form a reservoir of infection. Although direct transmission of tuberculosis occurs between mammals, the low frequency of contact between different host species and abundant shedding of bacilli by infected animals suggests an infectious route via environmental contamination. Other intracellular pathogens that transmit via the environment deploy strategies to survive or exploit predation by environmental amoebae. To explore if M. bovis has this capability, we investigated its interactions with the soil and dung-dwelling amoeba, Dictyostelium discoideum. We demonstrated that M. bovis evades phagocytosis and destruction by D. discoideum and actively transits through the amoeba using the ESX-1 Type VII Secretion System as part of a programme of mechanisms, many of which have been co-opted as virulence factors in the mammalian host. This capacity of M. bovis to utilise an environmental stage between mammalian hosts may enhance its transmissibility. In addition, our data provide molecular evidence to support an evolutionary role for amoebae as training grounds for the pathogenic M. tuberculosis complex.
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Affiliation(s)
- Rachel E Butler
- Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, GU2 7XH, UK
| | - Alex A Smith
- Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, GU2 7XH, UK
| | - Tom A Mendum
- Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, GU2 7XH, UK
| | - Aneesh Chandran
- Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, GU2 7XH, UK
| | - Huihai Wu
- Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, GU2 7XH, UK
| | - Louise Lefrançois
- Department of Biochemistry, Science II, University of Geneva, 30 quai Ernest-Ansermet, Geneva, Switzerland
| | - Mark Chambers
- School of Veterinary Medicine, University of Surrey, Guildford, Surrey, GU2 7AL, UK
| | - Thierry Soldati
- Department of Biochemistry, Science II, University of Geneva, 30 quai Ernest-Ansermet, Geneva, Switzerland
| | - Graham R Stewart
- Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, Surrey, GU2 7XH, UK.
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29
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Gautam US, Mehra S, Kumari P, Alvarez X, Niu T, Tyagi JS, Kaushal D. Mycobacterium tuberculosis sensor kinase DosS modulates the autophagosome in a DosR-independent manner. Commun Biol 2019; 2:349. [PMID: 31552302 PMCID: PMC6754383 DOI: 10.1038/s42003-019-0594-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2018] [Accepted: 09/03/2019] [Indexed: 01/03/2023] Open
Abstract
Dormancy is a key characteristic of the intracellular life-cycle of Mtb. The importance of sensor kinase DosS in mycobacteria are attributed in part to our current findings that DosS is required for both persistence and full virulence of Mtb. Here we show that DosS is also required for optimal replication in macrophages and involved in the suppression of TNF-α and autophagy pathways. Silencing of these pathways during the infection process restored full virulence in MtbΔdosS mutant. Notably, a mutant of the response regulator DosR did not exhibit the attenuation in macrophages, suggesting that DosS can function independently of DosR. We identified four DosS targets in Mtb genome; Rv0440, Rv2859c, Rv0994, and Rv0260c. These genes encode functions related to hypoxia adaptation, which are not directly controlled by DosR, e.g., protein recycling and chaperoning, biosynthesis of molybdenum cofactor and nitrogen metabolism. Our results strongly suggest a DosR-independent role for DosS in Mtb.
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Affiliation(s)
- Uma S. Gautam
- Tulane National Primate Research Center, Covington, LA 70433 USA
- Present Address: Duke Human Vaccine Institute, Duke University School of Medicine, 909 S. LaSalle St., Durham, NC 27710 USA
| | - Smriti Mehra
- Tulane National Primate Research Center, Covington, LA 70433 USA
- Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803 USA
- Center for Experimental Infectious Diseases Research, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803 USA
| | - Priyanka Kumari
- All India Institute of Medical Sciences, New Delhi, 110029 India
| | - Xavier Alvarez
- Tulane National Primate Research Center, Covington, LA 70433 USA
| | - Tianhua Niu
- Department of Biochemistry, Tulane University School of Medicine, New Orleans, 70112 LA USA
| | - Jaya S. Tyagi
- All India Institute of Medical Sciences, New Delhi, 110029 India
- Centre for Bio-design and Diagnostics, Translational Health Science and Technology Institute Faridabad, Haryana, 121001 India
| | - Deepak Kaushal
- Tulane National Primate Research Center, Covington, LA 70433 USA
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, 70112 LA USA
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30
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Stamm CE, Pasko BL, Chaisavaneeyakorn S, Franco LH, Nair VR, Weigele BA, Alto NM, Shiloh MU. Screening Mycobacterium tuberculosis Secreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector. mSphere 2019; 4:e00354-19. [PMID: 31167949 PMCID: PMC6553557 DOI: 10.1128/msphere.00354-19] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Accepted: 05/19/2019] [Indexed: 02/07/2023] Open
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.
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Affiliation(s)
- Chelsea E Stamm
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Breanna L Pasko
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Sujittra Chaisavaneeyakorn
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Luis H Franco
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Vidhya R Nair
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Bethany A Weigele
- Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Neal M Alto
- Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Michael U Shiloh
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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31
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The role of low molecular weight thiols in Mycobacterium tuberculosis. Tuberculosis (Edinb) 2019; 116:44-55. [PMID: 31153518 DOI: 10.1016/j.tube.2019.04.003] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2018] [Revised: 04/16/2019] [Accepted: 04/22/2019] [Indexed: 02/06/2023]
Abstract
Low molecular weight (LMW) thiols are molecules with a functional sulfhydryl group that enable them to detoxify reactive oxygen species, reactive nitrogen species and other free radicals. Their roles range from their ability to modulate the immune system to their ability to prevent damage of biological molecules such as DNA and proteins by protecting against oxidative, nitrosative and acidic stress. LMW thiols are synthesized and found in both eukaryotes and prokaryotes. Due to their beneficial role to both eukaryotes and prokaryotes, their specific functions need to be elucidated, most especially in pathogenic prokaryotes such as Mycobacterium tuberculosis (M.tb), in order to provide a rationale for targeting their biosynthesis for drug development. Ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC) are LMW thiols that have been shown to interplay to protect M.tb against cellular stress. Though ERG, MSH and GGC seem to have overlapping functions, studies are gradually revealing their unique physiological roles. Understanding their unique physiological role during the course of tuberculosis (TB) infection, would pave the way for the development of drugs that target their biosynthetic pathway. This review identifies the knowledge gap in the unique physiological roles of LMW thiols and proposes their mechanistic roles based on previous studies. In addition, it gives an update on identified inhibitors of their biosynthetic enzymes.
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Slayden RA, Dawson CC, Cummings JE. Toxin-antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis. Pathog Dis 2018; 76:4969681. [PMID: 29788125 DOI: 10.1093/femspd/fty039] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Accepted: 05/01/2018] [Indexed: 11/14/2022] Open
Abstract
There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.
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Affiliation(s)
- Richard A Slayden
- Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-0922, USA
| | - Clinton C Dawson
- Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-0922, USA
| | - Jason E Cummings
- Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-0922, USA
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Segura-Cerda CA, Aceves-Sánchez MDJ, Marquina-Castillo B, Mata-Espinoza D, Barrios-Payán J, Vega-Domínguez PJ, Pedroza-Roldán C, Bravo-Madrigal J, Vallejo-Cardona AA, Hernández-Pando R, Flores-Valdez MA. Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence. Vaccine 2018; 36:2069-2078. [PMID: 29550192 DOI: 10.1016/j.vaccine.2018.03.014] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Revised: 02/22/2018] [Accepted: 03/07/2018] [Indexed: 12/20/2022]
Abstract
Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1β, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4+ and T CD8+ activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses.
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Affiliation(s)
- Cristian Alfredo Segura-Cerda
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico
| | - Michel de Jesús Aceves-Sánchez
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico
| | - Brenda Marquina-Castillo
- Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico
| | - Dulce Mata-Espinoza
- Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico
| | - Jorge Barrios-Payán
- Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico
| | - Perla Jazmín Vega-Domínguez
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico
| | - César Pedroza-Roldán
- Departamento de Medicina Veterinaria, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Zapopan, Mexico
| | - Jorge Bravo-Madrigal
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico
| | - Alba Adriana Vallejo-Cardona
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico
| | - Rogelio Hernández-Pando
- Departamento de Patología Experimental, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico
| | - Mario Alberto Flores-Valdez
- Biotecnología Médica y Farmacéutica, Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico.
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Wilburn KM, Fieweger RA, VanderVen BC. Cholesterol and fatty acids grease the wheels of Mycobacterium tuberculosis pathogenesis. Pathog Dis 2018; 76:4931720. [PMID: 29718271 PMCID: PMC6251666 DOI: 10.1093/femspd/fty021] [Citation(s) in RCA: 117] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2017] [Accepted: 03/06/2018] [Indexed: 01/23/2023] Open
Abstract
Tuberculosis is a distinctive disease in which the causative agent, Mycobacterium tuberculosis, can persist in humans for decades by avoiding clearance from host immunity. During infection, M. tuberculosis maintains viability by extracting and utilizing essential nutrients from the host, and this is a prerequisite for all of the pathogenic activities that are deployed by the bacterium. In particular, M. tuberculosis preferentially acquires and metabolizes host-derived lipids (fatty acids and cholesterol), and the bacterium utilizes these substrates to cause and maintain disease. In this review, we discuss our current understanding of lipid utilization by M. tuberculosis, and we describe how these pathways promote pathogenesis to fuel metabolic processes in the bacillus. Finally, we highlight weaknesses in these pathways that potentially can be targeted for drug discovery.
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Affiliation(s)
- Kaley M Wilburn
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA
| | - Rachael A Fieweger
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA
| | - Brian C VanderVen
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA
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Cardenal-Muñoz E, Barisch C, Lefrançois LH, López-Jiménez AT, Soldati T. When Dicty Met Myco, a (Not So) Romantic Story about One Amoeba and Its Intracellular Pathogen. Front Cell Infect Microbiol 2018; 7:529. [PMID: 29376033 PMCID: PMC5767268 DOI: 10.3389/fcimb.2017.00529] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2017] [Accepted: 12/18/2017] [Indexed: 01/06/2023] Open
Abstract
In recent years, Dictyostelium discoideum has become an important model organism to study the cell biology of professional phagocytes. This amoeba not only shares many molecular features with mammalian macrophages, but most of its fundamental signal transduction pathways are conserved in humans. The broad range of existing genetic and biochemical tools, together with its suitability for cell culture and live microscopy, make D. discoideum an ideal and versatile laboratory organism. In this review, we focus on the use of D. discoideum as a phagocyte model for the study of mycobacterial infections, in particular Mycobacterium marinum. We look in detail at the intracellular cycle of M. marinum, from its uptake by D. discoideum to its active or passive egress into the extracellular medium. In addition, we describe the molecular mechanisms that both the mycobacterial invader and the amoeboid host have developed to fight against each other, and compare and contrast with those developed by mammalian phagocytes. Finally, we introduce the methods and specific tools that have been used so far to monitor the D. discoideum-M. marinum interaction.
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Affiliation(s)
- Elena Cardenal-Muñoz
- Department of Biochemistry, Sciences II, Faculty of Sciences, University of Geneva, Geneva, Switzerland
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36
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Lerner TR, Queval CJ, Fearns A, Repnik U, Griffiths G, Gutierrez MG. Phthiocerol dimycocerosates promote access to the cytosol and intracellular burden of Mycobacterium tuberculosis in lymphatic endothelial cells. BMC Biol 2018; 16:1. [PMID: 29325545 PMCID: PMC5795283 DOI: 10.1186/s12915-017-0471-6] [Citation(s) in RCA: 58] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Accepted: 12/13/2017] [Indexed: 01/09/2023] Open
Abstract
Background Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs. Results Analysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden. Conclusions PDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans.
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Affiliation(s)
- Thomas R Lerner
- The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
| | | | - Antony Fearns
- The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
| | - Urska Repnik
- Department of Biosciences, University of Oslo, Blindernveien 31, 0371, Oslo, Norway
| | - Gareth Griffiths
- Department of Biosciences, University of Oslo, Blindernveien 31, 0371, Oslo, Norway
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Identification of additional loci associated with antibody response to Mycobacterium avium ssp. Paratuberculosis in cattle by GSEA-SNP analysis. Mamm Genome 2017; 28:520-527. [PMID: 28864882 DOI: 10.1007/s00335-017-9714-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2017] [Accepted: 08/27/2017] [Indexed: 10/18/2022]
Abstract
Mycobacterium avium subsp. paratuberculosis: (MAP) causes a contagious chronic infection results in Johne's disease in a wide range of animal species, including cattle. Several genome-wide association studies (GWAS) have been carried out to identify loci putatively associated with MAP susceptibility by testing each marker separately and identifying SNPs that show a significant association with the phenotype, while SNP with modest effects are usually ignored. The objective of this study was to identify modest-effect genes associated with MAP susceptibility using a pathway-based approach. The Illumina BovineSNP50 BeadChip was used to genotype 966 Holstein cows, 483 positive and 483 negative for antibody response to MAP, data were then analyzed using novel SNP-based Gene Set Enrichment Analysis (GSEA-SNP) and validated with Adaptive Rank Truncated Product methodology. An allele-based test was carried out to estimate the statistical association for each marker with the phenotype, subsequently SNPs were mapped to the closest genes, considering for each gene the single variant with the highest value within a window of 50 kb, then pathway-statistics were tested using the GSEA-SNP method. The GO biological process "embryogenesis and morphogenesis" was most highly associated with antibody response to MAP. Within this pathway, five genes code for proteins which play a role in the immune defense relevant to response to bacterial infection. The immune response genes identified would not have been considered using a standard GWAS, thus demonstrating that the pathway approach can extend the interpretation of genome-wide association analyses and identify additional candidate genes for target traits.
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Kumar G, Shankar H, Sharma D, Sharma P, Bisht D, Katoch VM, Joshi B. Proteomics of Culture Filtrate of Prevalent Mycobacterium tuberculosis Strains: 2D-PAGE Map and MALDI-TOF/MS Analysis. SLAS DISCOVERY 2017; 22:1142-1149. [PMID: 28683213 DOI: 10.1177/2472555217717639] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Although diverse efforts have been done to identify biomarkers for control of tuberculosis using laboratory strain Mycobacterium tuberculosis H37Rv, the disease still poses a threat to mankind. There are many emerging M. tuberculosis strains, and proteomic profiling of these strains might be important to find out potential targets for diagnosis and/or prevention of tuberculosis. We evaluated the comparative proteomic profiling of culture filtrate (CF) proteins from prevalent M. tuberculosis strains (Central Asian or Delhi type; CAS1_Del, East African-Indian; EAI-3 and Beijing family) by 2D polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. As a result, we could identify 12 CF proteins (Rv0066c, Rv1310, Rv3375, Rv1415, Rv0567, Rv1886c, Rv3803c, Rv3804c, Rv2031c, Rv1038c, Rv2809, and Rv1911c), which were consistently increased in all prevalent M. tuberculosis strains, and interestingly, two CF proteins (Rv2809, Rv1911c) were identified with unknown functions. Consistent increased intensity of these proteins suggests their critical role for survival of prevalent M. tuberculosis isolates, and some of these proteins may also have potential as diagnostic and vaccine candidates for tuberculosis, which needs to be further explored by immunological analysis.
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Affiliation(s)
- Gavish Kumar
- Department of Immunology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Taj Ganj, Agra, Uttar Pradesh, India.,Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, Sri Aurobindo Marg, New Delhi, India
| | - Hari Shankar
- Department of Immunology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Taj Ganj, Agra, Uttar Pradesh, India
| | - Divakar Sharma
- Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Taj Ganj, Agra, Uttar Pradesh, India
| | - Prashant Sharma
- Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Taj Ganj, Agra, Uttar Pradesh, India
| | - Deepa Bisht
- Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Taj Ganj, Agra, Uttar Pradesh, India
| | - Vishwa M Katoch
- Department of Health Research (Ministry of Health and Family Welfare), Indian Council of Medical Research, V. Ramalingaswami Bhawan, Ansari Nagar, New Delhi, India
| | - Beenu Joshi
- Department of Immunology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (ICMR), Taj Ganj, Agra, Uttar Pradesh, India
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39
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Sundaramurthy V, Korf H, Singla A, Scherr N, Nguyen L, Ferrari G, Landmann R, Huygen K, Pieters J. Survival of Mycobacterium tuberculosis and Mycobacterium bovis BCG in lysosomes in vivo. Microbes Infect 2017; 19:515-526. [PMID: 28689009 DOI: 10.1016/j.micinf.2017.06.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2016] [Revised: 03/31/2017] [Accepted: 06/27/2017] [Indexed: 12/24/2022]
Abstract
Mycobacterium tuberculosis is one of the most successful pathogens known, having infected more than a third of the global population. An important strategy for intracellular survival of pathogenic mycobacteria relies on their capacity to resist delivery to lysosomes, instead surviving within macrophage phagosomes. Several factors of both mycobacterial and host origin have been implicated in this process. However, whether or not this strategy is employed in vivo is not clear. Here we show that in vivo, following intravenous infection, M. tuberculosis and Mycobacterium bovis BCG initially survived by resisting lysosomal transfer. However, after prolonged infection the bacteria were transferred to lysosomes yet continued to proliferate. A M. bovis BCG mutant lacking protein kinase G (PknG), that cannot avoid lysosomal transfer and is readily cleared in vitro, was found to survive and proliferate in vivo. The ability to survive and proliferate in lysosomal organelles in vivo was found to be due to an altered host environment rather than changes in the inherent ability of the bacteria to arrest phagosome maturation. Thus, within an infected host, both M. tuberculosis and M. bovis BCG adapts to infection-specific host responses. These results are important to understand the pathology of tuberculosis and may have implications for the development of effective strategies to combat tuberculosis.
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Affiliation(s)
| | - Hannelie Korf
- Scientific Institute of Public Health (WIV-ISP (Site Ukkel)), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
| | - Ashima Singla
- National Center for Biological Sciences, GKVK, Bellary Road, Bengaluru, India
| | - Nicole Scherr
- Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, Switzerland
| | - Liem Nguyen
- Department of Molecular Biology and Microbiology, Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Ave, LC 4860, Cleveland, OH, USA
| | - Giorgio Ferrari
- Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, Switzerland
| | - Regine Landmann
- Department of Biomedicine, University Hospital, Hebelstrasse 20, 4056, Basel, Switzerland
| | - Kris Huygen
- Scientific Institute of Public Health (WIV-ISP (Site Ukkel)), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
| | - Jean Pieters
- Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, Switzerland
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Mikheecheva NE, Zaychikova MV, Melerzanov AV, Danilenko VN. A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages. Genome Biol Evol 2017; 9:887-899. [PMID: 28338924 PMCID: PMC5381574 DOI: 10.1093/gbe/evx053] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/09/2017] [Indexed: 02/06/2023] Open
Abstract
Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin–antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed.
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Affiliation(s)
- Natalya E Mikheecheva
- Vavilov Institute of General Genetics, Moscow, Russia.,Moscow Institute of Physics and Technology, Dolgoprudny, Russia
| | | | | | - Valery N Danilenko
- Vavilov Institute of General Genetics, Moscow, Russia.,Scientific Research Center of Biotechnology of Antibiotics BIOAN, Moscow, Russia
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41
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Perkowski EF, Zulauf KE, Weerakoon D, Hayden JD, Ioerger TR, Oreper D, Gomez SM, Sacchettini JC, Braunstein M. The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection. mBio 2017; 8:e00333-17. [PMID: 28442606 PMCID: PMC5405230 DOI: 10.1128/mbio.00333-17] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2017] [Accepted: 04/04/2017] [Indexed: 12/12/2022] Open
Abstract
Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivotechnology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions.IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.
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Affiliation(s)
- E F Perkowski
- Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - K E Zulauf
- Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - D Weerakoon
- Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - J D Hayden
- Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - T R Ioerger
- Department of Computer Science and Engineering, Texas A&M University, College Station, Texas, USA
| | - D Oreper
- Joint Department of Biomedical Engineering at UNC-Chapel Hill and NC State University, Chapel Hill, North Carolina, USA
| | - S M Gomez
- Joint Department of Biomedical Engineering at UNC-Chapel Hill and NC State University, Chapel Hill, North Carolina, USA
| | - J C Sacchettini
- Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA
| | - M Braunstein
- Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
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42
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Mycobacterium llatzerense, a waterborne Mycobacterium, that resists phagocytosis by Acanthamoeba castellanii. Sci Rep 2017; 7:46270. [PMID: 28393860 PMCID: PMC5385496 DOI: 10.1038/srep46270] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Accepted: 03/10/2017] [Indexed: 12/20/2022] Open
Abstract
Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria. Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii. Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis, were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns.
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Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance. Sci Rep 2017; 7:45120. [PMID: 28345623 PMCID: PMC5366871 DOI: 10.1038/srep45120] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2016] [Accepted: 01/27/2017] [Indexed: 12/26/2022] Open
Abstract
Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.
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Augenstreich J, Arbues A, Simeone R, Haanappel E, Wegener A, Sayes F, Le Chevalier F, Chalut C, Malaga W, Guilhot C, Brosch R, Astarie-Dequeker C. ESX-1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis. Cell Microbiol 2017; 19. [PMID: 28095608 DOI: 10.1111/cmi.12726] [Citation(s) in RCA: 147] [Impact Index Per Article: 18.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2016] [Revised: 01/12/2017] [Accepted: 01/14/2017] [Indexed: 12/20/2022]
Abstract
Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT-6). This small protein, which is secreted by the type VII secretion system ESX-1 (T7SS/ESX-1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock-out or knock-in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX-1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.
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Affiliation(s)
- Jacques Augenstreich
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Ainhoa Arbues
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Roxane Simeone
- Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France
| | - Evert Haanappel
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Alice Wegener
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Fadel Sayes
- Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France
| | - Fabien Le Chevalier
- Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France
| | - Christian Chalut
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Wladimir Malaga
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Christophe Guilhot
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
| | - Roland Brosch
- Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France
| | - Catherine Astarie-Dequeker
- Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS-Université de Toulouse (UPS), Toulouse, France
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Delogu G, Brennan MJ, Manganelli R. PE and PPE Genes: A Tale of Conservation and Diversity. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1019:191-207. [PMID: 29116636 DOI: 10.1007/978-3-319-64371-7_10] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
PE and PPE are two large families of proteins typical of mycobacteria whose structural genes in the Mycobacterium tuberculosis complex (MTBC) occupy about 7% of the total genome. The most ancestral PE and PPE proteins are expressed by genes that belong to the same operon and in most cases are found inserted in the esx clusters, encoding a type VII secretion system. Duplication and expansion of pe and ppe genes, coupled with intragenomic and intergenomic recombination events, led to the emergence of the polymorphic pe_pgrs and ppe_mptr genes in the MTBC genome. The role and function of these proteins, and particularly of the polymorphic subfamilies, remains elusive, although it is widely accepted that PE and PPE proteins may represent a specialized collection used by MTBC to interact with the complex host immune system of mammals. In this chapter, we summarize what has been discovered since the identification of these genes in 1998, focusing on M. tuberculosis genetic variability, host-pathogen interaction and TB pathogenesis.
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Affiliation(s)
- Giovanni Delogu
- Institute of Microbiology, Università Cattolica del Sacro Cuore, Largo A. Gemelli, 8, 00168, Rome, Italy.
| | | | - Riccardo Manganelli
- Department of Molecular Medicine, University of Padua, Via A. Gabelli, 63, 35121, Padua, Italy
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Devasundaram S, Gopalan A, Das SD, Raja A. Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen's Specific Memory T Cells in Healthy Household Contacts. Front Microbiol 2016; 7:1275. [PMID: 27667981 PMCID: PMC5017210 DOI: 10.3389/fmicb.2016.01275] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2016] [Accepted: 08/02/2016] [Indexed: 01/08/2023] Open
Abstract
In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4(+) central memory T cells in the circulation of HHC compared to PTB (p < 0.005 for ESAT-6 and p < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during in vitro hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms.
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Affiliation(s)
- Santhi Devasundaram
- Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India
| | - Akilandeswari Gopalan
- Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India
| | - Sulochana D Das
- Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India
| | - Alamelu Raja
- Department of Immunology, National Institute for Research in Tuberculosis (ICMR) Chennai, India
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A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany. Int J Med Microbiol 2016; 306:495-503. [PMID: 27481640 DOI: 10.1016/j.ijmm.2016.07.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Revised: 06/06/2016] [Accepted: 07/17/2016] [Indexed: 11/21/2022] Open
Abstract
Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A-H (sized 6.2-73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.
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Ates LS, van der Woude AD, Bestebroer J, van Stempvoort G, Musters RJP, Garcia-Vallejo JJ, Picavet DI, Weerd RVD, Maletta M, Kuijl CP, van der Wel NN, Bitter W. The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10. PLoS Pathog 2016; 12:e1005696. [PMID: 27280885 PMCID: PMC4900558 DOI: 10.1371/journal.ppat.1005696] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2015] [Accepted: 05/20/2016] [Indexed: 11/18/2022] Open
Abstract
Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.
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Affiliation(s)
- Louis S Ates
- Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands
| | - Aniek D van der Woude
- Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands.,Department of Molecular Microbiology, VU University, Amsterdam, the Netherlands
| | - Jovanka Bestebroer
- Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands
| | | | - René J P Musters
- Department of Physiology and Cardiology, VU University Medical Center, Amsterdam, the Netherlands
| | - Juan J Garcia-Vallejo
- Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, the Netherlands
| | - Daisy I Picavet
- Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam the Netherlands
| | - Robert van de Weerd
- Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands
| | | | - Coenraad P Kuijl
- Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands
| | - Nicole N van der Wel
- Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam the Netherlands
| | - Wilbert Bitter
- Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands.,Department of Molecular Microbiology, VU University, Amsterdam, the Netherlands
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Pawar K, Sharbati J, Einspanier R, Sharbati S. Mycobacterium bovis BCG Interferes with miR-3619-5p Control of Cathepsin S in the Process of Autophagy. Front Cell Infect Microbiol 2016; 6:27. [PMID: 27014637 PMCID: PMC4783571 DOI: 10.3389/fcimb.2016.00027] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2015] [Accepted: 02/22/2016] [Indexed: 12/14/2022] Open
Abstract
Main survival mechanism of pathogenic mycobacteria is to escape inimical phagolysosomal environment inside the macrophages. Many efforts have been made to unravel the molecular mechanisms behind this process. However, little is known about the involvement of microRNAs (miRNAs) in the regulation of phagolysosomal biosynthesis and maturation. Based on a bottom up approach, we searched for miRNAs that were involved in phagolysosomal processing events in the course of mycobacterial infection of macrophages. After infecting THP-1 derived macrophages with viable and heat killed Mycobacterium bovis BCG (BCG), early time points were identified after co-localization studies of the phagosomal marker protein LAMP1 and BCG. Differences in LAMP1 localization on the phagosomes of both groups were observed at 30 min and 4 h. After in silico based pre-selection of miRNAs, expression analysis at the identified time points revealed down-regulation of three miRNAs: miR-3619-5p, miR-637, and miR-324-3p. Consequently, most likely targets were predicted that were supposed to be mutually regulated by these three studied miRNAs. The lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4) were up-regulated and were considered to be connected to lysosomal trafficking and autophagy. Interaction studies verified the regulation of CTSS by miR-3619-5p. Down-regulation of CTSS by ectopic miR-3619-5p as well as its specific knockdown by siRNA affected the process of autophagy in THP-1 derived macrophages.
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Affiliation(s)
- Kamlesh Pawar
- Department of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin Berlin, Germany
| | - Jutta Sharbati
- Department of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin Berlin, Germany
| | - Ralf Einspanier
- Department of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin Berlin, Germany
| | - Soroush Sharbati
- Department of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin Berlin, Germany
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Perkowski EF, Miller BK, McCann JR, Sullivan JT, Malik S, Allen IC, Godfrey V, Hayden JD, Braunstein M. An orphaned Mce-associated membrane protein of Mycobacterium tuberculosis is a virulence factor that stabilizes Mce transporters. Mol Microbiol 2016; 100:90-107. [PMID: 26712165 DOI: 10.1111/mmi.13303] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/03/2015] [Indexed: 12/17/2022]
Abstract
Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mam protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.
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Affiliation(s)
| | - Brittany K Miller
- Department of Microbiology and Immunology, University of North Carolina
| | - Jessica R McCann
- Department of Microbiology and Immunology, University of North Carolina
| | | | - Seidu Malik
- Department of Microbiology and Immunology, University of North Carolina
| | - Irving Coy Allen
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine
| | - Virginia Godfrey
- Department of Pathology and Laboratory Medicine, University of North Carolina
| | - Jennifer D Hayden
- Department of Microbiology and Immunology, University of North Carolina
| | - Miriam Braunstein
- Department of Microbiology and Immunology, University of North Carolina
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