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Singh J, Pradhan P, Kataria A, Sinha S, Ehtesham NZ, Monk PN, Hasnain SE. Conservation of Putative Liquid-Liquid Phase Separating Proteins in Multiple Drug-Resistant Mycobacterium tuberculosis: Role in Host-Pathogen Interactions? ACS Infect Dis 2025; 11:1034-1041. [PMID: 40183374 DOI: 10.1021/acsinfecdis.4c00722] [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] [Indexed: 04/05/2025]
Abstract
We observed a high proportion of proteins in pathogenic Mycobacterium species that can potentially undergo liquid-liquid phase separation (LLPS) mediated biomolecular condensate formation, compared to nonpathogenic species. These proteins mainly include the PE-PPE and PE-PGRS families of proteins that have nucleic acid and protein-protein binding functions, typical of LLPS proteins. We also mapped identified LLPS proteins in M. tuberculosis (M.tb) drug-resistant databases PubMLST and TBProfiler, based upon the WHO 2023 catalogue of resistance-associated mutations. High sequence conservation of LLPS-associated proteins in various multiple drug-resistant M.tb isolates points to their potentially important role in virulence and host-pathogen interactions during pathogenic evolution. This analysis provides a perspective on the role of protein phase separation in the evaluation of M.tb pathogenesis and offers avenues for future research aimed at developing innovative strategies to combat M.tb infection.
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Affiliation(s)
- Jasdeep Singh
- Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80210, United States
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi 110016, India
| | - Prashant Pradhan
- Laboratory of Nuclear Organization, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Research, Department of Obstetrics and Gynecology, Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9096, United States
| | - Arti Kataria
- Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana 59840, United States
| | - Sanjeev Sinha
- Department of Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
| | - Nasreen Z Ehtesham
- Department of Life Science, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh 201310, India
| | - Peter N Monk
- Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2TN, U.K
| | - Seyed E Hasnain
- Department of Life Science, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh 201310, India
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi 110016, India
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Ehtram A, Shariq M, Quadir N, Jamal S, Pichipalli M, Zarin S, Sheikh JA, Ehtesham NZ, Hasnain SE. Deciphering the functional roles of PE18 and PPE26 proteins in modulating Mycobacterium tuberculosis pathogenesis and immune response. Front Immunol 2025; 16:1517822. [PMID: 39949767 PMCID: PMC11821933 DOI: 10.3389/fimmu.2025.1517822] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2024] [Accepted: 01/07/2025] [Indexed: 02/16/2025] Open
Abstract
Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality worldwide. A crucial factor in Mtb's virulence is the ESX-5 secretion system, which transports PE/PPE proteins such as PE18 and PPE26. These proteins modulate host-pathogen interactions, immune responses, and intracellular survival mechanisms. Despite their importance, the roles and molecular interactions of PE18 and PPE26 in Mtb pathogenesis require further investigation. Methods We explored the roles of PE18 and PPE26 using recombinant Mycobacterium smegmatis (Msmeg) as a model organism. Protein-protein interactions were analyzed biochemically to identify partners within the ESX-5 secretion system, including EspG5 and other PE/PPE proteins. Subcellular localization of these proteins was assessed via cell fractionation studies. Functional assays, including in vitro cytokine production and antigen presentation studies, were performed using TLR2/Myd88 knockout and wild-type macrophages. In vivo experiments were conducted to assess effector T-cell activation and intracellular survival. Mechanistic insights into endosome-phagosome maturation and actin cytoskeleton dynamics were obtained through fluorescence microscopy. Results Our biochemical analyses confirmed interactions between PE18/PPE26, PE18/PPE27, PE19/PPE25, and EspG5/PPE, highlighting their involvement in ESX-5-mediated secretion. Cell fractionation studies revealed that PE/PPE proteins predominantly localize to the cell wall, with PE18 also secreted extracellularly. In vitro and in vivo experiments demonstrated that PE18 and PPE26 activate cytokine production and antigen presentation via TLR2/Myd88-dependent signaling pathways, inducing robust effector memory T-cell responses. Recombinant Msmeg expressing PE18, PPE26, or their combination exhibited enhanced intracellular survival by disrupting endosome-phagosome maturation, likely through interference with actin cytoskeletal organization. Discussion Our findings elucidate the pivotal roles of PE18 and PPE26 in Mtb pathogenesis, emphasizing their contributions to immune modulation and intracellular persistence. The observed disruption of actin dynamics and endosome-phagosome maturation underscores a novel mechanism by which Mtb evades host defenses. The ability of PE18 and PPE26 to induce effector T-cell responses highlights their potential as targets for host-directed therapies or vaccine development against TB. Further studies focusing on their structure-function relationships and interactions with host proteins could accelerate the development of innovative therapeutic strategies.
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Affiliation(s)
- Aquib Ehtram
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India
| | - Mohd Shariq
- GITAM School of Science, Gandhi Institute of Technology and Management (GITAM) University, Hyderabad, Telangana, India
| | - Neha Quadir
- Inflammation Biology and Cell Signaling Laboratory, ICMR-National Institute of Pathology, New Delhi, India
- Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Salma Jamal
- Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Manjunath Pichipalli
- Inflammation Biology and Cell Signaling Laboratory, ICMR-National Institute of Pathology, New Delhi, India
| | - Sheeba Zarin
- Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
- Department of Life Science, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | | | - Nasreen Z. Ehtesham
- Department of Life Science, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Seyed E. Hasnain
- Department of Life Science, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India
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Wang P, Zhang G, Jiang L, Zhang S, Gao W, Wu Z, Li Y. Development of a Vaccine Candidate Based on Surface-Displayed Particles of Mycobacterium tuberculosis from the MTB39A Protein. Int J Mol Sci 2025; 26:797. [PMID: 39859511 PMCID: PMC11766116 DOI: 10.3390/ijms26020797] [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: 11/21/2024] [Revised: 01/01/2025] [Accepted: 01/14/2025] [Indexed: 01/27/2025] Open
Abstract
Tuberculosis (TB), a human and animal disease caused by Mycobacterium tuberculosis (M.tb), has the highest global mortality rate after coronavirus disease 2019 (COVID-19) and poses a major public health threat globally. Since 1890, vaccine candidates for various forms of TB have been developed for different age groups, but these vaccine candidates have not provided intended protection in adolescents and adults in clinical trials. To help prevent and control the spread of TB, the development of a safe and effective TB vaccine is imperative. The MTB39A protein and the molecular adjuvant MTB32C protein were expressed by an insect-baculovirus expression system, and the recombinant baculovirus surface-displayed particles were evaluated for their immunogenicity in BALB/c mice and calves. The results showed that the rvAc-71CA/rvAc-MTB39A recombinant baculovirus surface-displayed particles exhibited good immunogenicity in mice and calves and could be further developed as potential candidates.
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Affiliation(s)
- Pu Wang
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
| | - Gang Zhang
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
| | - Lingling Jiang
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
| | - Sinong Zhang
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
| | - Weifeng Gao
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
| | - Zhiwei Wu
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
- Center for Public Health Research, Medical School, Nanjing University, Nanjing 210023, China
| | - Yong Li
- Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, School of Life Sciences, Ningxia University, Yinchuan 750021, China; (P.W.); (G.Z.); (L.J.); (S.Z.); (W.G.)
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Wu F, Yang B, Xiao Y, Ren LL, Chen HY, Hu XL, Pan YY, Chen YS, Li HR. psk1 virulence gene-induced pulmonary and systemic tuberculosis in a young woman with normal immune function: A case report. World J Clin Cases 2024; 12:6826-6833. [PMID: 39687648 PMCID: PMC11525909 DOI: 10.12998/wjcc.v12.i35.6826] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 07/02/2024] [Accepted: 07/23/2024] [Indexed: 10/24/2024] Open
Abstract
BACKGROUND Tuberculosis is a chronic infectious disease and an important public health problem. Despite progress in controlling tuberculosis, the incidence of tuberculosis in China is still very high, with 895000 new cases annually. This case report describes the investigation of a case of severe disseminated tuberculosis in a young adult with normal immune function, conducted to ascertain why a Mycobacterium tuberculosis (M. tuberculosis) strain caused such severe disease. CASE SUMMARY A previously healthy 28-year-old woman presented to our hospital with a 1-month history of fever and fatigue. She was diagnosed with severe disseminated pulmonary tuberculosis, spinal tuberculosis with paravertebral abscesses, and tuberculous meningitis. M. tuberculosis was isolated from bronchoalveolar lavage fluid. She was treated with standard antituberculous therapy and underwent debridement, bone graft, and internal fixation surgery for spinal tuberculosis. She responded to therapy and regained her ability to walk following the surgery. We analysed the whole-genome sequence of the strain and designated it BLM-A21. Additional M. tuberculosis genomes were selected from the Virulence Factor Database (http://www.mgc.ac.cn/cgi-bin/VFs/genus.cgi?Genus=Mycobacterium) for comparison. An evolutionary tree of the BLM-A21 strain was built using PhyML maximum likelihood software. Further gene analysis revealed that, except for the pks1 gene, BLM-A21 had similar virulence genes to the CDC 1551 and H37Rv strains, which have lower dissemination. CONCLUSION We speculate that the pks1 virulence gene in BLM-A21 may be the key virulence gene responsible for the widespread dissemination of M. tuberculosis infection in this previously healthy adult with normal immune function.
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Affiliation(s)
- Fan Wu
- Department of Respiratory and Critical Care Medicine, Shengli Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, Fujian Province, China
| | - Bin Yang
- NHC Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
| | - Yan Xiao
- NHC Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
| | - Li-Li Ren
- NHC Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
| | - Hong-Yi Chen
- Emergency Department, Shengli Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, Fujian Province, China
| | - Xin-Lan Hu
- Clinical Microbiology Laboratory, Shengli Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, Fujian Province, China
| | - Yan-Yu Pan
- Infection Department, The 900th Hospital of the PLA Joint Support Force, Fuzhou 350001, Fujian Province, China
| | - Yu-Sheng Chen
- Department of Respiratory and Critical Care Medicine, Shengli Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, Fujian Province, China
| | - Hong-Ru Li
- Department of Infectious Diseases, Shengli Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou 350001, Fujian Province, China
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Malik AA, Shariq M, Sheikh JA, Jaiswal U, Fayaz H, Shrivastava G, Ehtesham NZ, Hasnain SE. Mechanisms of immune evasion by Mycobacterium tuberculosis: the impact of T7SS and cell wall lipids on host defenses. Crit Rev Biochem Mol Biol 2024; 59:310-336. [PMID: 39378051 DOI: 10.1080/10409238.2024.2411264] [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: 07/09/2024] [Revised: 09/21/2024] [Accepted: 09/27/2024] [Indexed: 11/14/2024]
Abstract
Mycobacterium tuberculosis (M. tb) is one of the most successful human pathogens, causing a severe and widespread infectious disease. The frequent emergence of multidrug-resistant (MDR) strains has exacerbated this public health crisis, particularly in underdeveloped regions. M. tb employs a sophisticated array of virulence factors to subvert host immune responses, both innate and adaptive. It utilizes the early secretory antigenic target (ESAT6) secretion system 1 (ESX-1) type VII secretion system (T7SS) and cell wall lipids to disrupt phagosomal integrity, inhibiting phagosome maturation, and fusion with lysosomes. Although host cells activate mechanisms such as ubiquitin (Ub), Ub-ligase, and cyclic GMP-AMP synthase-stimulator of interferon genes 1 (CGAS-STING1)-mediated autophagy to inhibit M. tb survival within macrophages, the pathogen counteracts these defenses with its own virulence factors, thereby inhibiting autophagy and dampening host-directed responses. T7SSs are critical for transporting proteins across the complex mycobacterial cell envelope, performing essential functions, including metabolite uptake, immune evasion, and conjugation. T7SS substrates fall into two main families: ESAT-6 system proteins, which are found in both Firmicutes and Actinobacteria, and proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) proteins, which are unique to mycobacteria. Recent studies have highlighted the significance of T7SSs in mycobacterial growth, virulence, and pathogenesis. Understanding the mechanisms governing T7SSs could pave the way for novel therapeutic strategies to combat mycobacterial diseases, including tuberculosis (TB).
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Affiliation(s)
- Asrar Ahmad Malik
- Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Mohd Shariq
- GITAM School of Science, GITAM University, Rudraram, Telangana, India
| | - Javaid Ahmad Sheikh
- Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, Hamdard Nagar, New Delhi, India
| | - Udyeshita Jaiswal
- Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, Hamdard Nagar, New Delhi, India
| | - Haleema Fayaz
- Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Gauri Shrivastava
- Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Nasreen Z Ehtesham
- Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Seyed E Hasnain
- Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi (IIT-D), Hauz Khas, New Delhi, India
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Yan L, Lai HY, Leung TCN, Cheng HF, Chen X, Tsui SKW, Ngai SM, Au SWN. PE/PPE Proteome and ESX-5 Substrate Spectrum in Mycobacterium marinum. Int J Mol Sci 2024; 25:9550. [PMID: 39273496 PMCID: PMC11395111 DOI: 10.3390/ijms25179550] [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: 07/13/2024] [Revised: 08/05/2024] [Accepted: 08/07/2024] [Indexed: 09/15/2024] Open
Abstract
PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.
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Affiliation(s)
- Lili Yan
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Hiu Ying Lai
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Thomas Chun Ning Leung
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Hiu Fu Cheng
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Xin Chen
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Stephen Kwok Wing Tsui
- School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Sai Ming Ngai
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
| | - Shannon Wing Ngor Au
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, China
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Fang WW, Kong XL, Yang JY, Tao NN, Li YM, Wang TT, Li YY, Han QL, Zhang YZ, Hu JJ, Li HC, Liu Y. PE/PPE mutations in the transmission of Mycobacterium tuberculosis in China revealed by whole genome sequencing. BMC Microbiol 2024; 24:206. [PMID: 38858614 PMCID: PMC11163795 DOI: 10.1186/s12866-024-03352-y] [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: 11/08/2023] [Accepted: 05/26/2024] [Indexed: 06/12/2024] Open
Abstract
OBJECTIVE This study aims to examine the impact of PE/PPE gene mutations on the transmission of Mycobacterium tuberculosis (M. tuberculosis) in China. METHODS We collected the whole genome sequencing (WGS) data of 3202 M. tuberculosis isolates in China from 2007 to 2018 and investigated the clustering of strains from different lineages. To evaluate the potential role of PE/PPE gene mutations in the dissemination of the pathogen, we employed homoplastic analysis to detect homoplastic single nucleotide polymorphisms (SNPs) within these gene regions. Subsequently, logistic regression analysis was conducted to analyze the statistical association. RESULTS Based on nationwide M. tuberculosis WGS data, it has been observed that the majority of the M. tuberculosis burden in China is caused by lineage 2 strains, followed by lineage 4. Lineage 2 exhibited a higher number of transmission clusters, totaling 446 clusters, of which 77 were cross-regional clusters. Conversely, there were only 52 transmission clusters in lineage 4, of which 9 were cross-regional clusters. In the analysis of lineage 2 isolates, regression results showed that 4 specific gene mutations, PE4 (position 190,394; c.46G > A), PE_PGRS10 (839,194; c.744 A > G), PE16 (1,607,005; c.620T > G) and PE_PGRS44 (2,921,883; c.333 C > A), were significantly associated with the transmission of M. tuberculosis. Mutations of PE_PGRS10 (839,334; c.884 A > G), PE_PGRS11 (847,613; c.1455G > C), PE_PGRS47 (3,054,724; c.811 A > G) and PPE66 (4,189,930; c.303G > C) exhibited significant associations with the cross-regional clusters. A total of 13 mutation positions showed a positive correlation with clustering size, indicating a positive association. For lineage 4 strains, no mutations were found to enhance transmission, but 2 mutation sites were identified as risk factors for cross-regional clusters. These included PE_PGRS4 (338,100; c.974 A > G) and PPE13 (976,897; c.1307 A > C). CONCLUSION Our results indicate that some PE/PPE gene mutations can increase the risk of M. tuberculosis transmission, which might provide a basis for controlling the spread of tuberculosis.
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Affiliation(s)
- Wei-Wei Fang
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, People's Republic of China
| | - Xiang-Long Kong
- Shandong Artificial Intelligence Institute, Qilu University of Technology & Shandong Academy of Sciences, Jinan, Shandong, PR China
| | - Jie-Yu Yang
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, People's Republic of China
| | - Ning-Ning Tao
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
| | - Ya-Meng Li
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong University of Traditional Chinese Medicine, Jinan, Shandong, PR China
| | - Ting-Ting Wang
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
| | - Ying-Ying Li
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong University of Traditional Chinese Medicine, Jinan, Shandong, PR China
| | - Qi-Lin Han
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, People's Republic of China
| | - Yu-Zhen Zhang
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, People's Republic of China
| | - Jin-Jiang Hu
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
- Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, People's Republic of China
| | - Huai-Chen Li
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China
| | - Yao Liu
- Department of Respiratory and Critical Care Medicine, Shandong Provincial Hospital, Shandong First Medical University, Jinan, Shandong, 250021, PR China.
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Manjunath P, Ahmad J, Samal J, Rani A, Sheikh JA, Zarin S, Ahuja Y, Alam A, Hasnain SE, Ehtesham NZ. Expression of a unique M. tuberculosis DNA MTase Rv1509 in M. smegmatis alters the gene expression pattern and enhances virulence. Front Microbiol 2024; 15:1344857. [PMID: 38803374 PMCID: PMC11129820 DOI: 10.3389/fmicb.2024.1344857] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Accepted: 04/15/2024] [Indexed: 05/29/2024] Open
Abstract
Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.
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Affiliation(s)
- P. Manjunath
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India
- Department of Biotechnology, Jamia Hamdard, New Delhi, India
| | - Javeed Ahmad
- Molecular Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States
| | - Jasmine Samal
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India
| | - Anshu Rani
- Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India
| | | | - Sheeba Zarin
- Department of Biotechnology, Jamia Hamdard, New Delhi, India
- Department of Life Science, Sharda School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Yashika Ahuja
- Department of Life Science, Sharda School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Anwar Alam
- Department of Biotechnology, Sharda School of Engineering Sciences and Technology, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Seyed E. Hasnain
- Department of Life Science, Sharda School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, New Delhi, India
| | - Nasreen Z. Ehtesham
- Department of Life Science, Sharda School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
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Negrete-Paz AM, Vázquez-Marrufo G, Gutiérrez-Moraga A, Vázquez-Garcidueñas MS. Pangenome Reconstruction of Mycobacterium tuberculosis as a Guide to Reveal Genomic Features Associated with Strain Clinical Phenotype. Microorganisms 2023; 11:1495. [PMID: 37374997 DOI: 10.3390/microorganisms11061495] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2023] [Revised: 05/31/2023] [Accepted: 06/02/2023] [Indexed: 06/29/2023] Open
Abstract
Tuberculosis (TB) is one of the leading causes of human deaths worldwide caused by infectious diseases. TB infection by Mycobacterium tuberculosis can occur in the lungs, causing pulmonary tuberculosis (PTB), or in any other organ of the body, resulting in extrapulmonary tuberculosis (EPTB). There is no consensus on the genetic determinants of this pathogen that may contribute to EPTB. In this study, we constructed the M. tuberculosis pangenome and used it as a tool to seek genomic signatures associated with the clinical presentation of TB based on its accessory genome differences. The analysis carried out in the present study includes the raw reads of 490 M. tuberculosis genomes (PTB n = 245, EPTB n = 245) retrieved from public databases that were assembled, as well as ten genomes from Mexican strains (PTB n = 5, EPTB n = 5) that were sequenced and assembled. All genomes were annotated and then used to construct the pangenome with Roary and Panaroo. The pangenome obtained using Roary consisted of 2231 core genes and 3729 accessory genes. On the other hand, the pangenome resulting from Panaroo consisted of 2130 core genes and 5598 accessory genes. Associations between the distribution of accessory genes and the PTB/EPTB phenotypes were examined using the Scoary and Pyseer tools. Both tools found a significant association between the hspR, plcD, Rv2550c, pe_pgrs5, pe_pgrs25, and pe_pgrs57 genes and the PTB genotype. In contrast, the deletion of the aceA, esxR, plcA, and ppe50 genes was significantly associated with the EPTB phenotype. Rv1759c and Rv3740 were found to be associated with the PTB phenotype according to Scoary; however, these associations were not observed when using Pyseer. The robustness of the constructed pangenome and the gene-phenotype associations is supported by several factors, including the analysis of a large number of genomes, the inclusion of the same number of PTB/EPTB genomes, and the reproducibility of results thanks to the different bioinformatic tools used. Such characteristics surpass most of previous M. tuberculosis pangenomes. Thus, it can be inferred that the deletion of these genes can lead to changes in the processes involved in stress response and fatty acid metabolism, conferring phenotypic advantages associated with pulmonary or extrapulmonary presentation of TB. This study represents the first attempt to use the pangenome to seek gene-phenotype associations in M. tuberculosis.
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Affiliation(s)
- Andrea Monserrat Negrete-Paz
- División de Estudios de Posgrado, Facultad de Ciencias Médicas y Biológicas "Dr. Ignacio Chávez", Universidad Michoacana de San Nicolás de Hidalgo, Morelia 58020, Michoacán, Mexico
- Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Tarímbaro 58893, Michoacán, Mexico
| | - Gerardo Vázquez-Marrufo
- Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Tarímbaro 58893, Michoacán, Mexico
| | - Ana Gutiérrez-Moraga
- Instituto de Ciencias Biomédicas, Vicerrectoría de Investigación y Doctorados, Universidad Autónoma de Chile, Santiago 7500912, Chile
| | - Ma Soledad Vázquez-Garcidueñas
- División de Estudios de Posgrado, Facultad de Ciencias Médicas y Biológicas "Dr. Ignacio Chávez", Universidad Michoacana de San Nicolás de Hidalgo, Morelia 58020, Michoacán, Mexico
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10
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Li W, Yan Z, Zhang N, Zhang Z, Xiang X. Novel role of PE_PGRS47 in the alteration of mycobacterial cell wall integrity and drug resistance. Arch Microbiol 2023; 205:174. [PMID: 37022460 DOI: 10.1007/s00203-023-03515-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 03/11/2023] [Accepted: 03/23/2023] [Indexed: 04/07/2023]
Abstract
The proline-glutamic acid and proline-proline-glutamic acid (PE/PPE) family of proteins is widespread in pathogenic mycobacteria and plays different roles in mycobacterial physiology. While several PE/PPE family proteins have been studied, the exact function of most PE/PPE proteins in the physiology of Mycobacterium tuberculosis (Mtb) remains unknown. PE_PGRS47 belongs to the PE/PPE family of proteins reported to help Mtb evade protective host immune responses. In this study, we demonstrate a novel role of PE_PGRS47. Heterologous expression of the pe_pgrs47 gene in a non-pathogenic Mycobacterium smegmatis, intrinsically deficient of PE_PGRS protein, exhibits modulated colony morphology and cell wall lipid profile leading to a marked susceptibility to multiple antibiotics and environmental stressors. Using ethidium bromide/Nile red uptake assays, Mycobacterium smegmatis expressing PE_PGRS47 showed higher cell wall permeability than the control strain. Overall, these data suggested that PE_PGRS47 is cell surface exposed and influences cell wall integrity and the formation of mycobacterial colonies, ultimately potentiating the efficacy of lethal stresses against mycobacteria.
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Affiliation(s)
- Wu Li
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, 641100, Sichuan, People's Republic of China.
| | - Zifei Yan
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, 641100, Sichuan, People's Republic of China
| | - Nan Zhang
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, 641100, Sichuan, People's Republic of China
| | - Zhiyong Zhang
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, 641100, Sichuan, People's Republic of China
| | - Xiaohong Xiang
- School of Pharmacy, Chongqing Medical and Pharmaceutical College, Chongqing, 401331, People's Republic of China.
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11
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Mendonca LE, Pernet E, Khan N, Sanz J, Kaufmann E, Downey J, Grant A, Orlova M, Schurr E, Krawczyk C, Jones RG, Barreiro LB, Divangahi M. Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection. Front Immunol 2023; 13:1044592. [PMID: 36776396 PMCID: PMC9910175 DOI: 10.3389/fimmu.2022.1044592] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Accepted: 11/21/2022] [Indexed: 01/28/2023] Open
Abstract
Pulmonary macrophages have two distinct ontogenies: long-lived embryonically-seeded alveolar macrophages (AM) and bone marrow-derived macrophages (BMDM). Here, we show that after infection with a virulent strain of Mycobacterium tuberculosis (H37Rv), primary murine AM exhibit a unique transcriptomic signature characterized by metabolic reprogramming distinct from conventional BMDM. In contrast to BMDM, AM failed to shift from oxidative phosphorylation (OXPHOS) to glycolysis and consequently were unable to control infection with an avirulent strain (H37Ra). Importantly, healthy human AM infected with H37Ra equally demonstrated diminished energetics, recapitulating our observation in the murine model system. However, the results from seahorse showed that the shift towards glycolysis in both AM and BMDM was inhibited by H37Rv. We further demonstrated that pharmacological (e.g. metformin or the iron chelator desferrioxamine) reprogramming of AM towards glycolysis reduced necrosis and enhanced AM capacity to control H37Rv growth. Together, our results indicate that the unique bioenergetics of AM renders these cells a perfect target for Mtb survival and that metabolic reprogramming may be a viable host targeted therapy against TB.
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Affiliation(s)
- Laura E. Mendonca
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada
| | - Erwan Pernet
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada
| | - Nargis Khan
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada
| | - Joaquin Sanz
- Institute for Biocomputation and Physics of Complex Systems (BIFI) for Biocomputation and Physics of Complex Systems and Department of Theoretical Physics, University of Zaragoza, Zaragoza, Spain
| | - Eva Kaufmann
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada
| | - Jeffrey Downey
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada
| | - Alexandre Grant
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada
| | - Marianna Orlova
- McGill International TB Centre, Montreal, QC, Canada,Department of Medicine and Human Genetics, McGill University. Program in Infectious Diseases and Immunity in Global Health, The Research Institute of the McGill University Health Centre, Montreal, QC, Canada
| | - Erwin Schurr
- McGill International TB Centre, Montreal, QC, Canada,Department of Medicine and Human Genetics, McGill University. Program in Infectious Diseases and Immunity in Global Health, The Research Institute of the McGill University Health Centre, Montreal, QC, Canada
| | - Connie Krawczyk
- Department of Physiology, Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada,VanAndel Institute, Center for Cancer and Cell Biology, Grand Rapids, MI, United States
| | - Russell G. Jones
- Department of Physiology, Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada,VanAndel Institute, Center for Cancer and Cell Biology, Grand Rapids, MI, United States
| | - Luis B. Barreiro
- McGill International TB Centre, Montreal, QC, Canada,Department of Genetics, Centre hospitalier de l'Université (CHU) Sainte-Justine Research Center, Montreal, QC, Canada,University of Chicago, Department of Medicine, Section of Genetic Medicine, Chicago, IL, United States
| | - Maziar Divangahi
- The Research Institute of the McGill University Health Centre, Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology and,McGill International TB Centre, Montreal, QC, Canada,*Correspondence: Maziar Divangahi,
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12
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Shariq M, Quadir N, Alam A, Zarin S, Sheikh JA, Sharma N, Samal J, Ahmad U, Kumari I, Hasnain SE, Ehtesham NZ. The exploitation of host autophagy and ubiquitin machinery by Mycobacterium tuberculosis in shaping immune responses and host defense during infection. Autophagy 2023; 19:3-23. [PMID: 35000542 PMCID: PMC9809970 DOI: 10.1080/15548627.2021.2021495] [Citation(s) in RCA: 53] [Impact Index Per Article: 26.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
Intracellular pathogens have evolved various efficient molecular armaments to subvert innate defenses. Cellular ubiquitination, a normal physiological process to maintain homeostasis, is emerging one such exploited mechanism. Ubiquitin (Ub), a small protein modifier, is conjugated to diverse protein substrates to regulate many functions. Structurally diverse linkages of poly-Ub to target proteins allow enormous functional diversity with specificity being governed by evolutionarily conserved enzymes (E3-Ub ligases). The Ub-binding domain (UBD) and LC3-interacting region (LIR) are critical features of macroautophagy/autophagy receptors that recognize Ub-conjugated on protein substrates. Emerging evidence suggests that E3-Ub ligases unexpectedly protect against intracellular pathogens by tagging poly-Ub on their surfaces and targeting them to phagophores. Two E3-Ub ligases, PRKN and SMURF1, provide immunity against Mycobacterium tuberculosis (M. tb). Both enzymes conjugate K63 and K48-linked poly-Ub to M. tb for successful delivery to phagophores. Intriguingly, M. tb exploits virulence factors to effectively dampen host-directed autophagy utilizing diverse mechanisms. Autophagy receptors contain LIR-motifs that interact with conserved Atg8-family proteins to modulate phagophore biogenesis and fusion to the lysosome. Intracellular pathogens have evolved a vast repertoire of virulence effectors to subdue host-immunity via hijacking the host ubiquitination process. This review highlights the xenophagy-mediated clearance of M. tb involving host E3-Ub ligases and counter-strategy of autophagy inhibition by M. tb using virulence factors. The role of Ub-binding receptors and their mode of autophagy regulation is also explained. We also discuss the co-opting and utilization of the host Ub system by M. tb for its survival and virulence.Abbreviations: APC: anaphase promoting complex/cyclosome; ATG5: autophagy related 5; BCG: bacille Calmette-Guerin; C2: Ca2+-binding motif; CALCOCO2: calcium binding and coiled-coil domain 2; CUE: coupling of ubiquitin conjugation to ER degradation domains; DUB: deubiquitinating enzyme; GABARAP: GABA type A receptor-associated protein; HECT: homologous to the E6-AP carboxyl terminus; IBR: in-between-ring fingers; IFN: interferon; IL1B: interleukin 1 beta; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LGALS: galectin; LIR: LC3-interacting region; MAPK11/p38: mitogen-activated protein kinase 11; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK8/JNK: mitogen-activated protein kinase 8; MHC-II: major histocompatibility complex-II; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB1/p50: nuclear factor kappa B subunit 1; OPTN: optineurin; PB1: phox and bem 1; PE/PPE: proline-glutamic acid/proline-proline-glutamic acid; PknG: serine/threonine-protein kinase PknG; PRKN: parkin RBR E3 ubiquitin protein ligase; RBR: RING-in between RING; RING: really interesting new gene; RNF166: RING finger protein 166; ROS: reactive oxygen species; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; UBA: ubiquitin-associated; UBAN: ubiquitin-binding domain in ABIN proteins and NEMO; UBD: ubiquitin-binding domain; UBL: ubiquitin-like; ULK1: unc-51 like autophagy activating kinase 1.
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Affiliation(s)
- Mohd Shariq
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India
| | - Neha Quadir
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India,Department of Molecular Medicine, Jamia Hamdard-Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Anwar Alam
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India
| | - Sheeba Zarin
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India,Department of Molecular Medicine, Jamia Hamdard-Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Javaid A. Sheikh
- Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India
| | - Neha Sharma
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India,Department of Molecular Medicine, Jamia Hamdard-Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Jasmine Samal
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India
| | - Uzair Ahmad
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India
| | - Indu Kumari
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India
| | - Seyed E. Hasnain
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi (IIT-D), New Delhi, India,Department of Life Science, School of Basic Sciences and Research, Sharda University, Greater Noida, India,Seyed E. Hasnain ; ; Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi (IIT-D), Hauz Khas, New Delhi 110 016, India
| | - Nasreen Z. Ehtesham
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology-ICMR, Ansari Nagar West, New Delhi, India,CONTACT Nasreen Z. Ehtesham ; ICMR-National Institute of Pathology, Ansari Nagar West, New Delhi110029, India
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13
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Sahoo S, Dehury B, Narang PK, Raina V, Misra N, Suar M. Comprehensive sequence and structure analysis of algal lipid catabolic enzyme Triacylglycerol lipase: an in silico study to vitalize the development of optimum engineered strains with high lipid productivity. J Biomol Struct Dyn 2022; 40:11989-12007. [PMID: 34415234 DOI: 10.1080/07391102.2021.1967194] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Microalgae as an alternative renewable resource for biofuel production have captured much significance. Nonetheless, its economic viability is a field of major concern for researchers. Unraveling the lipid catabolic pathway and gaining insights into the sequence-structural features of its primary functioning enzyme, Triacylglycerol lipase, will impart valuable information to target microalgae for augmented lipid content. In the present study, a genome-wide comparative study on putative Triacylglycerol lipase (TAGL) enzyme from algal species belonging to varied phylogenetic lineages was performed. The comprehensive sequence analysis revealed that TAGL comprises of three distinct conserved domains, such as, Patatin, Class III Lipase, and Abhydro_lipase, and also confirmed the ubiquitous presence of GXSXG motif in the sequences analyzed. In the absence of a crystal structure of algal TAGL till date, we developed the first 3D model of patatin domain of TAGL from an oleaginous microalga, Phaedactylum tricornutum, employing homology modeling, docking and molecular dynamic simulations methods. The domain-substrate complex having the low-ranking docking score revealed the binding of palmitic acid to the TAGL patatin domain surface with strong hydrogen bond interactions. The simulation results implied that the substrate-complexed patatin domain and the free enzyme adopted a more stable conformation after 40 ns. This is the first ever attempt to provide in-silico insights into the structural and dynamical insights on catalytic mechanism of the TAGL patatin domain. Subsequently, these findings aided our understanding on their structural stability, folding mechanism and protein-substrate interactions, which could be further utilized to design site-specific mutagenic experiments for engineering microalgal strains.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Susrita Sahoo
- School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India
| | - Budheswar Dehury
- Department of Chemistry, Technical University of Denmark, Lyngby, Denmark
| | - Parminder Kaur Narang
- School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India.,SGTB Khalsa College, Delhi University, Delhi, India
| | - Vishakha Raina
- School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India
| | - Namrata Misra
- School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India.,KIIT-Technology Business Incubator (KIIT-TBI), Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India
| | - Mrutyunjay Suar
- School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India.,KIIT-Technology Business Incubator (KIIT-TBI), Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, India
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14
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The Mycobacterium tuberculosis PE_PGRS Protein Family Acts as an Immunological Decoy to Subvert Host Immune Response. Int J Mol Sci 2022; 23:ijms23010525. [PMID: 35008950 PMCID: PMC8745494 DOI: 10.3390/ijms23010525] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2021] [Revised: 12/03/2021] [Accepted: 12/15/2021] [Indexed: 02/04/2023] Open
Abstract
Mycobacterium tuberculosis (M.tb) is a successful pathogen that can reside within the alveolar macrophages of the host and can survive in a latent stage. The pathogen has evolved and developed multiple strategies to resist the host immune responses. M.tb escapes from host macrophage through evasion or subversion of immune effector functions. M.tb genome codes for PE/PPE/PE_PGRS proteins, which are intrinsically disordered, redundant and antigenic in nature. These proteins perform multiple functions that intensify the virulence competence of M.tb majorly by modulating immune responses, thereby affecting immune mediated clearance of the pathogen. The highly repetitive, redundant and antigenic nature of PE/PPE/PE_PGRS proteins provide a critical edge over other M.tb proteins in terms of imparting a higher level of virulence and also as a decoy molecule that masks the effect of effector molecules, thereby modulating immuno-surveillance. An understanding of how these proteins subvert the host immunological machinery may add to the current knowledge about M.tb virulence and pathogenesis. This can help in redirecting our strategies for tackling M.tb infections.
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15
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PGRS Domain of Rv0297 of Mycobacterium tuberculosis Functions in A Calcium Dependent Manner. Int J Mol Sci 2021; 22:ijms22179390. [PMID: 34502303 PMCID: PMC8430768 DOI: 10.3390/ijms22179390] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Revised: 03/12/2021] [Accepted: 03/24/2021] [Indexed: 01/04/2023] Open
Abstract
Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca2+ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca2+ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca2+ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca2+ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca2+ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca2+ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.
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Ehtram A, Shariq M, Ali S, Quadir N, Sheikh JA, Ahmad F, Sharma T, Ehtesham NZ, Hasnain SE. Teleological cooption of Mycobacterium tuberculosis PE/PPE proteins as porins: Role in molecular immigration and emigration. Int J Med Microbiol 2021; 311:151495. [PMID: 33730677 DOI: 10.1016/j.ijmm.2021.151495] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 02/01/2021] [Accepted: 03/05/2021] [Indexed: 01/09/2023] Open
Abstract
Permeation through bacterial cells for exchange or uptake of biomolecules and ions invariably depend upon the existence of pore-forming proteins (porins) in their outer membrane. Mycobacterium tuberculosis (M. tb) harbours one of the most rigid cell envelopes across bacterial genera and is devoid of the classical porins for solute transport across the cell membrane. Though canonical porins are incompatible with the evolution of permeability barrier, porin like activity has been reported from membrane preparations of pathogenic mycobacteria. This suggests a sophisticated transport mechanism that has been elusive until now, along with the protein family responsible for it. Recent evidence suggests that these slow-growing mycobacteria have co-opted some of PE/PPE family proteins as molecular transport channels, in place of porins, to facilitate uptake of nutrients required to thrive in the restrictive host environment. These reports advocate that PE/PPE proteins, due to their structural ability, have a potential role in importing small molecules to the cell's interior. This mechanism unveils how a successful pathogen overcomes its restrictive membrane's transport limitations for selective uptake of nutrients. If extrapolated to have a role in drug transport, these channels could help understand the emergence of drug resistance. Further, as these proteins are associated with the export of virulence factors, they can be exploited as novel drug targets. There remains, however, an interesting question that as the PE/PPE proteins can allow the 'import' of molecules from outside the cell, is the reverse transport also possible across the M. tb membrane. In this review, we have discussed recent evidence supporting PE/PPE's role as a specific transport channel for selective uptake of small molecule nutrients and, as possible molecular export machinery of M. tb. This newly discovered role as transmembrane channels demands further research on this enigmatic family of proteins to comprehend the pathomechanism of this very smart pathogen.
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Affiliation(s)
- Aquib Ehtram
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India
| | - Mohd Shariq
- ICMR-National Institute of Pathology, Ansari Nagar West, New Delhi, India
| | - Sabeeha Ali
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India
| | - Neha Quadir
- ICMR-National Institute of Pathology, Ansari Nagar West, New Delhi, India; Jamia Hamdard- Institute of Molecular Medicine, Jamia Hamdard, Hamdard Nagar, New Delhi, India
| | - Javaid A Sheikh
- Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, Hamdard Nagar, New Delhi, 110062, India
| | - Faraz Ahmad
- ICMR-National Institute of Pathology, Ansari Nagar West, New Delhi, India
| | - Tarina Sharma
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi, India; ICMR-National Institute of Pathology, Ansari Nagar West, New Delhi, India
| | - Nasreen Z Ehtesham
- ICMR-National Institute of Pathology, Ansari Nagar West, New Delhi, India.
| | - Seyed E Hasnain
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India; Dr Reddy's Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India.
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17
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Shariq M, Quadir N, Sharma N, Singh J, Sheikh JA, Khubaib M, Hasnain SE, Ehtesham NZ. Mycobacterium tuberculosis RipA Dampens TLR4-Mediated Host Protective Response Using a Multi-Pronged Approach Involving Autophagy, Apoptosis, Metabolic Repurposing, and Immune Modulation. Front Immunol 2021; 12:636644. [PMID: 33746976 PMCID: PMC7969667 DOI: 10.3389/fimmu.2021.636644] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Accepted: 02/03/2021] [Indexed: 12/26/2022] Open
Abstract
Reductive evolution has endowed Mycobacterium tuberculosis (M. tb) with moonlighting in protein functions. We demonstrate that RipA (Rv1477), a peptidoglycan hydrolase, activates the NFκB signaling pathway and elicits the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12, through the activation of an innate immune-receptor, toll-like receptor (TLR)4. RipA also induces an enhanced expression of macrophage activation markers MHC-II, CD80, and CD86, suggestive of M1 polarization. RipA harbors LC3 (Microtubule-associated protein 1A/1B-light chain 3) motifs known to be involved in autophagy regulation and indeed alters the levels of autophagy markers LC3BII and P62/SQSTM1 (Sequestosome-1), along with an increase in the ratio of P62/Beclin1, a hallmark of autophagy inhibition. The use of pharmacological agents, rapamycin and bafilomycin A1, reveals that RipA activates PI3K-AKT-mTORC1 signaling cascade that ultimately culminates in the inhibition of autophagy initiating kinase ULK1 (Unc-51 like autophagy activating kinase). This inhibition of autophagy translates into efficient intracellular survival, within macrophages, of recombinant Mycobacterium smegmatis expressing M. tb RipA. RipA, which also localizes into mitochondria, inhibits the production of oxidative phosphorylation enzymes to promote a Warburg-like phenotype in macrophages that favors bacterial replication. Furthermore, RipA also inhibited caspase-dependent programed cell death in macrophages, thus hindering an efficient innate antibacterial response. Collectively, our results highlight the role of an endopeptidase to create a permissive replication niche in host cells by inducing the repression of autophagy and apoptosis, along with metabolic reprogramming, and pointing to the role of RipA in disease pathogenesis.
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Affiliation(s)
- Mohd Shariq
- Indian Council of Medical Research-National Institute of Pathology, New Delhi, India
| | - Neha Quadir
- Indian Council of Medical Research-National Institute of Pathology, New Delhi, India.,Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Neha Sharma
- Indian Council of Medical Research-National Institute of Pathology, New Delhi, India.,Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Jasdeep Singh
- Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Javaid A Sheikh
- Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India
| | - Mohd Khubaib
- Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Seyed E Hasnain
- Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India.,Dr. Reddy's Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India.,Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi (IIT-D) Hauz Khas, New Delhi, India
| | - Nasreen Z Ehtesham
- Indian Council of Medical Research-National Institute of Pathology, New Delhi, India
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18
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Mallick Gupta A, Mandal S. Distribution of sigma factors delineates segregation of virulent and avirulent Mycobacterium. Arch Microbiol 2021; 203:1627-1640. [PMID: 33432378 DOI: 10.1007/s00203-020-02172-8] [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: 08/08/2020] [Revised: 12/02/2020] [Accepted: 12/27/2020] [Indexed: 11/26/2022]
Abstract
The genus Mycobacterium includes a wide range of species of both slow and rapid growth under major pathogens, opportunists, and saprophytes. The number and combination of sigma factors are extremely diversified among various species of Mycobacterium. The comparative genome analysis illustrates that SigC, SigD, SigG, SigH, SigK and SigI are dominant among the pathogens. Evolutionary analysis using Bayesian inference on 16S rRNA and MLST-based phylogeny using 14 housekeeping genes distinctly differentiate the slow-growing Mycobacterium from fast growers and segregate pathogens from opportunists and saprophytes. Based on the similarity coefficient upon the allotment of sigma factors in mycobacterial species through UPGMA dendrogram analysis, it is apparent that the pathogens are grouped separately following the similar trend observed from the evolutionary approach. Predominance of a set of sigma factors particularly the pathogenic Mycobacterium co-exists with the distribution of six well-known virulence factors of Mycobacterium (PhoP, PcaA, FbpA, Mce1B, KatG and PE_PGRS30). The pathogenicity responsible sigma factors elicit close resemblance with few notable characters of the known virulence factors. Thus the analysis renders that the distribution of sigma factors of different species of Mycobacterium can be a potential tool to predict their pathogenicity index.
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Affiliation(s)
- Aayatti Mallick Gupta
- Laboratory of Molecular Bacteriology, Department of Microbiology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India
| | - Sukhendu Mandal
- Laboratory of Molecular Bacteriology, Department of Microbiology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India.
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19
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Comprehensive analysis of protein acetyltransferases of human pathogen Mycobacterium tuberculosis. Biosci Rep 2020; 39:221456. [PMID: 31820790 PMCID: PMC6923341 DOI: 10.1042/bsr20191661] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 11/20/2019] [Accepted: 11/26/2019] [Indexed: 12/12/2022] Open
Abstract
Tuberculosis (TB), a leading infectious disease caused by Mycobacterium tuberculosis strain, takes four human lives every minute globally. Paucity of knowledge on M. tuberculosis virulence and antibiotic resistance is the major challenge for tuberculosis control. We have identified 47 acetyltransferases in the M. tuberculosis, which use diverse substrates including antibiotic, amino acids, and other chemical molecules. Through comparative analysis of the protein file of the virulent M. tuberculosis H37Rv strain and the avirulent M. tuberculosis H37Ra strain, we identified one acetyltransferase that shows significant variations with N-terminal deletion, possibly influencing its physicochemical properties. We also found that one acetyltransferase has three types of post-translation modifications (lysine acetylation, succinylation, and glutarylation). The genome context analysis showed that many acetyltransferases with their neighboring genes belong to one operon. By data mining from published transcriptional profiles of M. tuberculosis exposed to diverse treatments, we revealed that several acetyltransferases may be functional during M. tuberculosis infection. Insights obtained from the present study can potentially provide clues for developing novel TB therapeutic interventions.
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20
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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: 21] [Impact Index Per Article: 4.2] [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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21
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Sheikh JA, Ehtesham NZ, Hasnain SE. Revisiting BCG to control tuberculosis: mucosal delivery and delipidation? THE LANCET. INFECTIOUS DISEASES 2020; 20:272-273. [PMID: 32112751 DOI: 10.1016/s1473-3099(19)30702-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/20/2019] [Revised: 11/21/2019] [Accepted: 11/22/2019] [Indexed: 02/02/2023]
Affiliation(s)
- Javaid Ahmad Sheikh
- Department of Biotechnology, School of Chemical and Life Sciences, New Delhi, India
| | - Nasreen Zafar Ehtesham
- Inflammation Biology and Cell Signalling Laboratory, Indian Council of Medical Research National Institute of Pathology, New Delhi 110029, India.
| | - Seyed Ehtesham Hasnain
- Institute of Molecular Medicine, School of Interdisciplinary Sciences, New Delhi 110062, India; Dr Reddy's Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India; Robert Koch Institute, Berlin, Germany.
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22
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Sharma M, Zhang S, Niu L, Lewinsohn DM, Zhang X, Huang S. Mucosal-Associated Invariant T Cells Develop an Innate-Like Transcriptomic Program in Anti-mycobacterial Responses. Front Immunol 2020; 11:1136. [PMID: 32582206 PMCID: PMC7295940 DOI: 10.3389/fimmu.2020.01136] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2020] [Accepted: 05/11/2020] [Indexed: 12/12/2022] Open
Abstract
Conventional T cells exhibit a delayed response to the initial priming of peptide antigens presented by major histocompatibility complex (MHC) proteins. Unlike conventional T cells, mucosal-associated invariant T (MAIT) cells quickly respond to non-peptidic metabolite antigens presented by MHC-related protein 1 (MR1). To elucidate the MR1-dependent activation program of MAIT cells in response to mycobacterial infections, we determined the surface markers, transcriptomic profiles, and effector responses of activated human MAIT cells. Results revealed that mycobacterial-incubated antigen-presenting cells stimulated abundant human CD8+ MAIT cells to upregulate the co-expression of CD69 and CD26, as a combinatorial activation marker. Further transcriptomic analyses demonstrated that CD69+CD26++ CD8+MAIT cells highly expressed numerous genes for mediating anti-mycobacterial immune responses, including pro-inflammatory cytokines, cytolytic molecules, NK cell receptors, and transcription factors, in contrast to inactivated counterparts CD69+/−CD26+/− CD8+MAIT cells. Gene co-expression, enrichment, and pathway analyses yielded high statistical significance to strongly support that activated CD8+ MAIT cells shared gene expression and numerous pathways with NK and CD8+ T cells in activation, cytokine production, cytokine signaling, and effector functions. Flow cytometry detected that activated CD8+MAIT cells produced TNFα, IFNγ, and granulysin to inhibit mycobacterial growth and fight mycobacterial infection. Together, results strongly support that the combinatorial activation marker CD69+CD26++ labels the activated CD8+MAIT cells that develop an innate-like activation program in anti-mycobacterial immune responses. We speculate that the rapid production of anti-mycobacterial effector molecules facilitates MAIT cells to fight early mycobacterial infection in humans.
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Affiliation(s)
- Manju Sharma
- Division of Environmental Genetics and Molecular Toxicology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH, United States
| | - Shuangmin Zhang
- Division of Environmental Genetics and Molecular Toxicology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH, United States
| | - Liang Niu
- Division of Biostatistics and Bioinformatics, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH, United States
| | - David M Lewinsohn
- Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, OR, United States
| | - Xiang Zhang
- Division of Environmental Genetics and Molecular Toxicology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH, United States.,Genomics, Epigenomics and Sequencing Core, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH, United States
| | - Shouxiong Huang
- Division of Environmental Genetics and Molecular Toxicology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH, United States.,Immunobiology Graduate Program, Cincinnati Children's Hospital, Cincinnati, OH, United States
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23
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Ahmad J, Khubaib M, Sheikh JA, Pancsa R, Kumar S, Srinivasan A, Babu MM, Hasnain SE, Ehtesham NZ. Disorder-to-order transition in PE-PPE proteins of Mycobacterium tuberculosis augments the pro-pathogen immune response. FEBS Open Bio 2019; 10:70-85. [PMID: 31643141 PMCID: PMC6943233 DOI: 10.1002/2211-5463.12749] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Revised: 10/11/2019] [Accepted: 10/22/2019] [Indexed: 12/23/2022] Open
Abstract
A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host–pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine–alanine‐rich PE–PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE–PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host–pathogen interactions. As a case study, the secondary structure of PE35–PPE68 and PE32–PPE65 of the pathogenesis‐related RD1 and RD8 regions was analyzed through Fourier‐transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro‐pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder–order structural dynamics as a strategy to mount a pro‐pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution.
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Affiliation(s)
- Javeed Ahmad
- Inflammation Biology and Cell Signalling Laboratory, National Institute of Pathology, New Delhi, India.,Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
| | - Mohd Khubaib
- JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
| | - Javaid Ahmad Sheikh
- Inflammation Biology and Cell Signalling Laboratory, National Institute of Pathology, New Delhi, India.,Department of Biotechnology, Jamia Hamdard, New Delhi, India
| | - Rita Pancsa
- Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK
| | - Saroj Kumar
- Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
| | - Alagiri Srinivasan
- Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
| | - Mohan Madan Babu
- Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK
| | - Seyed E Hasnain
- JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India.,Dr. Reddy's Institute of Life Sciences, Hyderabad, India
| | - Nasreen Z Ehtesham
- Inflammation Biology and Cell Signalling Laboratory, National Institute of Pathology, New Delhi, India
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24
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Li W, Deng W, Xie J. Expression and regulatory networks of Mycobacterium tuberculosis PE/PPE family antigens. J Cell Physiol 2018; 234:7742-7751. [PMID: 30478834 DOI: 10.1002/jcp.27608] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2018] [Accepted: 09/21/2018] [Indexed: 01/06/2023]
Abstract
PE/PPE family antigens are distributed mainly in pathogenic mycobacteria and serve as potential antituberculosis (TB) vaccine components. Some PE/PPE family antigens can regulate the host innate immune response, interfere with macrophage activation and phagolysosome fusion, and serve as major sources of antigenic variation. PE/PPE antigens have been associated with mycobacteria pathogenesis; pe/ppe genes are mainly found in pathogenic mycobacteria and are differentially expressed between Mtb and Mycobacterium bovis. PE/PPE proteins were essential for the growth of Mtb, and PE/PPE proteins were differentially expressed under a variety of conditions. Multiple mycobacterial-virulence-related transcription factors, sigma factors, the global transcriptional regulation factor Lsr2, MprAB, and PhoPR two-component regulatory systems, and cyclic adenine monophosphate-dependent regulators, regulate the expression of PE/PPE family antigens. Multiple-scale integrative analysis revealed the expression and regulatory networks of PE/PPE family antigens underlying the virulence and pathogenesis of Mtb, providing important clues for the discovery of new anti-TB measures.
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Affiliation(s)
- Wu Li
- Key Laboratory of Regional Characteristic Agricultural Resources, College of Life Sciences, Neijiang Normal University, Neijiang, China
| | - Wanyan Deng
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
| | - Jianping Xie
- Institute of Modern Biopharmaceuticals, 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, Southwest University, Beibei, Chongqing, China
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25
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Barh D, Tiwari S, Kumavath RN, Ghosh P, Azevedo V. Linking common non-coding RNAs of human lung cancer and M. tuberculosis. Bioinformation 2018; 14:337-345. [PMID: 30237679 PMCID: PMC6137563 DOI: 10.6026/97320630014337] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2018] [Revised: 06/29/2018] [Accepted: 06/30/2018] [Indexed: 02/07/2023] Open
Abstract
Lung cancer and pulmonary tuberculosis caused by Mycobacterium are two major causes of deaths worldwide. Tuberculosis linked lung cancer is known. However, the precise molecular mechanism of Mycobacterium associated increased risk of lung cancer is not understood. We report 45 common human miRNAs deregulated in both pulmonary tuberculosis and lung cancer. We show that sRNA_1096 and sRNA_1414 from M. tuberculosis have sequence homology with human mir-21. Hence, the potential role of these three small non-coding RNAs in rifampicin resistance in pulmonary tuberculosis is implied. Further, the linking of sRNA_1096 and sRNA_1414 from M. tuberculosis with the host lung tumorigenesis is inferred. Nonetheless, further analysis and validation is required to associate these three non-coding RNAs with Mycobacterium associated increased risk of lung cancer.
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Affiliation(s)
- Debmalya Barh
- Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, West Bengal, India
- Laboratorio de Genetica Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciencias Biologicas (ICB), Universidade Federal de Minas Gerais, Pampulha, Belo Horizonte, Minas Gerais, Brazil
- Division of Bioinformatics and Computational Genomics, NITTE University Center for Science Education and Research (NUCSER), NITTE (Deemed to be University), Deralakatte, Mangaluru, Karnataka, India
| | - Sandeep Tiwari
- Laboratorio de Genetica Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciencias Biologicas (ICB), Universidade Federal de Minas Gerais, Pampulha, Belo Horizonte, Minas Gerais, Brazil
| | - Ranjith N. Kumavath
- Department of Genomic Science, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Periya (P.O) Kasaragod, Kerala-671316, India
| | - Preetam Ghosh
- Department of Computer Science, Virginia Commonwealth University, Virginia 23284, USA
| | - Vasco Azevedo
- Laboratorio de Genetica Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciencias Biologicas (ICB), Universidade Federal de Minas Gerais, Pampulha, Belo Horizonte, Minas Gerais, Brazil
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26
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The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4. mBio 2018; 9:mBio.01017-18. [PMID: 29921671 PMCID: PMC6016250 DOI: 10.1128/mbio.01017-18] [Citation(s) in RCA: 67] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism. Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease.
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27
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Singh G, Tripathi S, Shanker K, Sharma A. Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo, partially and fully metalated forms. J Biomol Struct Dyn 2018; 37:1520-1533. [PMID: 29624115 DOI: 10.1080/07391102.2018.1461688] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
Plants play an important role in the removal of excess heavy metals from soil and water. Medicinal plants can also have non-traditional use in phytoremediation technologies. Among the heavy metals, Cadmium (Cd) is the most abundant and readily taken up by the crop plants. Plant metallothioneins (MTs) are small proteins having cysteine-rich residues and appear to play key roles in metal homoeostasis. Plant metallothionein 2 (MT 2) from Coptis japonica (Gold-thread; CjMT 2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by 42 amino acids long linker region. In-silico analysis of MT 2 protein sequences of C. japonica was performed. In this study, ab initio methods were utilised for the prediction of three-dimensional structure of CjMT 2. After structure validation, heavy metal-binding sites were predicted for the selected modelled structures of CjMT 2. To obtain Cdi-CjMT 2 (i = 1-7), metalated complex individual docking experiments were performed. The stability of the metalated docked structures was assessed by molecular dynamics (MD) simulation studies. Our study showed that CjMT 2 binds up to 4 Cd2+ ions in two distinct domains: a N-terminal β-domain that binds to 2 Cd2+ ions and a C-terminal α-domain that binds with 2 Cd2+ ions. Our analysis revealed that Cys residues of alpha and beta domain and some residues of spacer region of CjMT 2 protein might be important for the cadmium interaction. MD simulation studies provided insight into metal-induced conformational changes and mechanism of metalation of CjMT 2, an intrinsically disordered protein. This study provides useful insights into mechanism of cadmium-type 2 metallothionein interaction.
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Affiliation(s)
- Garima Singh
- a Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants , Post Office CIMAP , Lucknow 226015 , India.,c Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad 201002 , India
| | - Shubhandra Tripathi
- a Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants , Post Office CIMAP , Lucknow 226015 , India
| | - Karuna Shanker
- b Chemical Science Division, CSIR-Central Institute of Medicinal and Aromatic Plants , Post Office CIMAP , Lucknow 226015 , India.,c Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad 201002 , India
| | - Ashok Sharma
- a Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants , Post Office CIMAP , Lucknow 226015 , India.,c Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad 201002 , India
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28
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Exploration of Mycobacterium tuberculosis structural proteome: An in-silico approach. J Theor Biol 2018; 439:14-23. [DOI: 10.1016/j.jtbi.2017.11.021] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Revised: 07/19/2017] [Accepted: 11/28/2017] [Indexed: 12/20/2022]
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29
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Leung KSS, Siu GKH, Tam KKG, To SWC, Rajwani R, Ho PL, Wong SSY, Zhao WW, Ma OCK, Yam WC. Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing. Front Cell Infect Microbiol 2017; 7:478. [PMID: 29188195 PMCID: PMC5694780 DOI: 10.3389/fcimb.2017.00478] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Accepted: 10/31/2017] [Indexed: 12/02/2022] Open
Abstract
Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.
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Affiliation(s)
- Kenneth Siu-Sing Leung
- Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong
| | - Gilman Kit-Hang Siu
- Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, Hong Kong
| | - Kingsley King-Gee Tam
- Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong
| | - Sabrina Wai-Chi To
- Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong
| | - Rahim Rajwani
- Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, Hong Kong
| | - Pak-Leung Ho
- Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong
| | - Samson Sai-Yin Wong
- Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong
| | - Wei W. Zhao
- KingMed Diagnostics, Science Park, Hong Kong, Hong Kong
| | | | - Wing-Cheong Yam
- Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong
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O'Toole RF, Gautam SS. Limitations of the Mycobacterium tuberculosis reference genome H37Rv in the detection of virulence-related loci. Genomics 2017; 109:471-474. [DOI: 10.1016/j.ygeno.2017.07.004] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2017] [Revised: 07/17/2017] [Accepted: 07/18/2017] [Indexed: 10/19/2022]
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31
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Chen X, Cheng HF, Zhou J, Chan CY, Lau KF, Tsui SKW, Au SWN. Structural basis of the PE-PPE protein interaction in Mycobacterium tuberculosis. J Biol Chem 2017; 292:16880-16890. [PMID: 28842489 DOI: 10.1074/jbc.m117.802645] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2017] [Revised: 08/16/2017] [Indexed: 11/06/2022] Open
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has developed multiple strategies to adapt to the human host. The five type VII secretion systems, ESX-1-5, direct the export of many virulence-promoting protein effectors across the complex mycobacterial cell wall. One class of ESX substrates is the PE-PPE family of proteins, which is unique to mycobacteria and essential for infection, antigenic variation, and host-pathogen interactions. The genome of Mtb encodes 168 PE-PPE proteins. Many of them are thought to be secreted through ESX-5 secretion system and to function in pairs. However, understanding of the specific pairing of PE-PPE proteins and their structure-function relationship is limited by the challenging purification of many PE-PPE proteins, and our knowledge of the PE-PPE interactions therefore has been restricted to the PE25-PPE41 pair and its complex with the ESX-5 secretion system chaperone EspG5. Here, we report the crystal structure of a new PE-PPE pair, PE8-PPE15, in complex with EspG5. Our structure revealed that the EspG5-binding sites on PPE15 are relatively conserved among Mtb PPE proteins, suggesting that EspG5-PPE15 represents a more typical model for EspG5-PPE interactions than EspG5-PPE41. A structural comparison with the PE25-PPE41 complex disclosed conformational changes in the four-helix bundle structure and a unique binding mode in the PE8-PPE15 pair. Moreover, homology-modeling and mutagenesis studies further delineated the molecular determinants of the specific PE-PPE interactions. These findings help develop an atomic algorithm of ESX-5 substrate recognition and PE-PPE pairing.
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Affiliation(s)
- Xin Chen
- From the Centre for Protein Science and Crystallography, School of Life Sciences
| | - Hiu-Fu Cheng
- From the Centre for Protein Science and Crystallography, School of Life Sciences
| | - Junwei Zhou
- From the Centre for Protein Science and Crystallography, School of Life Sciences
| | | | - Kwok-Fai Lau
- From the Centre for Protein Science and Crystallography, School of Life Sciences
| | | | - Shannon Wing-Ngor Au
- From the Centre for Protein Science and Crystallography, School of Life Sciences,
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32
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Selection and identification of specific glycoproteins and glycan biomarkers of macrophages involved in Mycobacterium tuberculosis infection. Tuberculosis (Edinb) 2017; 104:95-106. [DOI: 10.1016/j.tube.2017.03.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2016] [Revised: 02/18/2017] [Accepted: 03/28/2017] [Indexed: 01/06/2023]
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Elghraoui A, Modlin SJ, Valafar F. SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis. BMC Genomics 2017; 18:302. [PMID: 28415976 PMCID: PMC5393005 DOI: 10.1186/s12864-017-3687-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2016] [Accepted: 04/06/2017] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. RESULTS Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome. CONCLUSIONS Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.
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Affiliation(s)
- Afif Elghraoui
- Biological and Medical Informatics Research Center, San Diego State University, Campanile Drive, San Diego, 92182, USA
| | - Samuel J Modlin
- Biological and Medical Informatics Research Center, San Diego State University, Campanile Drive, San Diego, 92182, USA
| | - Faramarz Valafar
- Biological and Medical Informatics Research Center, San Diego State University, Campanile Drive, San Diego, 92182, USA.
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Díaz DP, Ocampo M, Varela Y, Curtidor H, Patarroyo MA, Patarroyo ME. Identifying and characterising PPE7 (Rv0354c) high activity binding peptides and their role in inhibiting cell invasion. Mol Cell Biochem 2017; 430:149-160. [PMID: 28205097 DOI: 10.1007/s11010-017-2962-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Accepted: 01/28/2017] [Indexed: 10/20/2022]
Abstract
This study was aimed at characterising the PPE7 protein from the PE/PPE protein family. The presence and transcription of the rv0354c gene in the Mycobacterium tuberculosis complex was determined and the subcellular localisation of the PPE7 protein on mycobacterial membrane was confirmed by immunoelectron microscope. Two peptides were identified as having high binding activity (HABPs) and were tested in vitro regarding the invasion of Mycobacterium tuberculosis H37Rv. HABP 39224 inhibited invasion in A549 epithelial cells and U937 macrophages by more than 50%, whilst HABP 39225 inhibited invasion by 40% in U937 cells. HABP 39224, located in the protein's C-terminal region, has a completely conserved amino acid sequence in M. tuberculosis complex species and could be selected as a base peptide when designing a subunit-based, anti-tuberculosis vaccine.
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Affiliation(s)
- Diana P Díaz
- Fundación Instituto de Inmunología de Colombia (FIDIC), 111321, Bogotá, Colombia.,Universidad del Rosario, 111321, Bogotá, Colombia
| | - Marisol Ocampo
- Fundación Instituto de Inmunología de Colombia (FIDIC), 111321, Bogotá, Colombia. .,Universidad del Rosario, 111321, Bogotá, Colombia.
| | - Yahson Varela
- Fundación Instituto de Inmunología de Colombia (FIDIC), 111321, Bogotá, Colombia.,Universidad del Rosario, 111321, Bogotá, Colombia
| | - Hernando Curtidor
- Fundación Instituto de Inmunología de Colombia (FIDIC), 111321, Bogotá, Colombia.,Universidad del Rosario, 111321, Bogotá, Colombia
| | - Manuel A Patarroyo
- Fundación Instituto de Inmunología de Colombia (FIDIC), 111321, Bogotá, Colombia.,Universidad del Rosario, 111321, Bogotá, Colombia
| | - Manuel E Patarroyo
- Fundación Instituto de Inmunología de Colombia (FIDIC), 111321, Bogotá, Colombia.,Universidad Nacional de Colombia, 11001, Bogotá, Colombia
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Chakraborty C, Bandyopadhyay S, Agoramoorthy G. India's Computational Biology Growth and Challenges. Interdiscip Sci 2016; 8:263-76. [PMID: 27465042 DOI: 10.1007/s12539-016-0179-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Revised: 09/08/2015] [Accepted: 09/08/2015] [Indexed: 11/30/2022]
Abstract
India's computational science is growing swiftly due to the outburst of internet and information technology services. The bioinformatics sector of India has been transforming rapidly by creating a competitive position in global bioinformatics market. Bioinformatics is widely used across India to address a wide range of biological issues. Recently, computational researchers and biologists are collaborating in projects such as database development, sequence analysis, genomic prospects and algorithm generations. In this paper, we have presented the Indian computational biology scenario highlighting bioinformatics-related educational activities, manpower development, internet boom, service industry, research activities, conferences and trainings undertaken by the corporate and government sectors. Nonetheless, this new field of science faces lots of challenges.
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Affiliation(s)
- Chiranjib Chakraborty
- Department of Bio-informatics, School of Computer and Information Sciences, Galgotias University, Greater Noida, India
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36
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Khubaib M, Sheikh JA, Pandey S, Srikanth B, Bhuwan M, Khan N, Hasnain SE, Ehtesham NZ. Mycobacterium tuberculosis Co-operonic PE32/PPE65 Proteins Alter Host Immune Responses by Hampering Th1 Response. Front Microbiol 2016; 7:719. [PMID: 27242739 PMCID: PMC4868851 DOI: 10.3389/fmicb.2016.00719] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Accepted: 04/29/2016] [Indexed: 02/04/2023] Open
Abstract
PE/PPE genes, present in cluster with ESAT-6 like genes, are suspected to have a role in antigenic variation and virulence of Mycobacterium tuberculosis. Their roles in immune evasion and immune modulation of host are also well documented. We present evidence that PE32/PPE65 present within the RD8 region are co-operonic, co-transcribed, and co-translated, and play role in modulating host immune responses. Experiments with macrophage cell lines revealed that this protein complex suppresses pro-inflammatory cytokines such as TNF-α and IL-6 whereas also inducing high expression of anti-inflammatory IL-10. Immunization of mice with these recombinant proteins dampens an effective Th1 response as evident from reduced frequency of IFN-γ and IL-2 producing CD4+ and CD8+ T cells. IgG sub-typing from serum of immunized mice revealed high levels of IgG1 when compared with IgG2a and IgG2b. Further IgG1/IgG2a ratio clearly demonstrated that the protein complex manipulates the host immune response favorable to the pathogen. Our results demonstrate that the co-transcribed and co-translated PE32 and PPE65 antigens are involved specifically in modulating anti-mycobacterial host immune response by hampering Th1 response.
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Affiliation(s)
- Mohd Khubaib
- Inflammation Biology and Cell Signaling Laboratory, National Institute of PathologyNew Delhi, India; Dr. Reddy's Institute of Life Sciences, University of Hyderabad CampusHyderabad, India
| | - Javaid A Sheikh
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology New Delhi, India
| | - Saurabh Pandey
- Inflammation Biology and Cell Signaling Laboratory, National Institute of PathologyNew Delhi, India; Dr. Reddy's Institute of Life Sciences, University of Hyderabad CampusHyderabad, India
| | - Battu Srikanth
- Department of Biotechnology, School of Life Sciences, University of Hyderabad Hyderabad, India
| | - Manish Bhuwan
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology New Delhi, India
| | - Nooruddin Khan
- Department of Biotechnology, School of Life Sciences, University of Hyderabad Hyderabad, India
| | - Seyed E Hasnain
- Dr. Reddy's Institute of Life Sciences, University of Hyderabad CampusHyderabad, India; Molecular Infection and Functional Biology Laboratory, Kusuma School of Biological Sciences, Indian Institute of TechnologyNew Delhi, India
| | - Nasreen Z Ehtesham
- Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology New Delhi, India
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37
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Grover S, Gupta P, Kahlon PS, Goyal S, Grover A, Dalal K, Sabeeha, Ehtesham NZ, Hasnain SE. Analyses of methyltransferases across the pathogenicity spectrum of different mycobacterial species point to an extremophile connection. MOLECULAR BIOSYSTEMS 2016; 12:1615-25. [PMID: 26983646 DOI: 10.1039/c5mb00810g] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Tuberculosis is a devastating disease, taking one human life every 20 seconds globally. We hypothesize that professional pathogens such as M.tb have acquired specific features that might assist in causing infection, persistence and transmissible pathology in their host. We have identified 121 methyltransferases (MTases) in the M.tb proteome, which use a variety of substrates - DNA, RNA, protein, intermediates of mycolic acid biosynthesis and other fatty acids - that are involved in cellular maintenance within the host. A comparative analysis of the proteome of the virulent strain H37Rv and the avirulent strain H37Ra identified 3 MTases, which displayed significant variations in terms of N-terminal extension/deletion and point mutations, possibly impacting various physicochemical properties. The cross-proteomic comparison of MTases of M.tb H37Rv with 15 different Mycobacterium species revealed the acquisition of novel MTases in a MTB complex as a function of evolution. Phylogenetic analysis revealed that these newly acquired MTases showed common roots with certain extremophiles such as halophilic and acidophilic organisms. Our results establish an evolutionary relationship of M.tb with halotolerant organisms and also the role of MTases of M.tb in withstanding the host osmotic stress, thereby pointing to their likely role in pathogenesis, virulence and niche adaptation.
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Affiliation(s)
- Sonam Grover
- Molecular Infection and Functional Biology Lab, Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India.
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Interaction of Mycobacterium tuberculosis Virulence Factor RipA with Chaperone MoxR1 Is Required for Transport through the TAT Secretion System. mBio 2016; 7:e02259. [PMID: 26933057 PMCID: PMC4810496 DOI: 10.1128/mbio.02259-15] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mycobacterium tuberculosis is a leading cause of death worldwide. The M. tuberculosis TAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. The M. tuberculosis secretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based on in silico analyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA in Mycobacterium smegmatis and copurification with MoxR1 further validated their interaction in vivo. Recombinant MoxR1 protein, expressed in Escherichia coli, displays ATP-enhanced chaperone activity. Secretion of recombinant RipA (rRipA) protein into the E. coli culture filtrate was not observed in the absence of RipA-MoxR interaction. Inhibition of this export system in M. tuberculosis, including the key players, will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing β-lactam antibiotics, opening up new candidates for drug repurposing. The virulence mechanism of mycobacteria is very complex. Broadly, the virulence factors can be classified as secretion factors, cell surface components, enzymes involved in cellular metabolism, and transcriptional regulators. The mycobacteria have evolved several mechanisms to secrete its proteins. Here, we have identified one of the virulence proteins of Mycobacterium tuberculosis, RipA, possessing peptidoglycan hydrolase activities secreted by the TAT secretion pathway. We also identified MoxR1 as a protein-protein interaction partner of RipA and demonstrated chaperone activity of this protein. We show that MoxR1-mediated folding is critical for the secretion of RipA within the TAT system. Inhibition of this export system in M. tuberculosis will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing β-lactam antibiotics, opening up new candidates for drug repurposing.
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39
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Sultana R, Tanneeru K, Kumar ABR, Guruprasad L. Prediction of Certain Well-Characterized Domains of Known Functions within the PE and PPE Proteins of Mycobacteria. PLoS One 2016; 11:e0146786. [PMID: 26891364 PMCID: PMC4758615 DOI: 10.1371/journal.pone.0146786] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2015] [Accepted: 12/22/2015] [Indexed: 11/18/2022] Open
Abstract
The PE and PPE protein family are unique to mycobacteria. Though the complete genome sequences for over 500 M. tuberculosis strains and mycobacterial species are available, few PE and PPE proteins have been structurally and functionally characterized. We have therefore used bioinformatics tools to characterize the structure and function of these proteins. We selected representative members of the PE and PPE protein family by phylogeny analysis and using structure-based sequence annotation identified ten well-characterized protein domains of known function. Some of these domains were observed to be common to all mycobacterial species and some were species specific.
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Affiliation(s)
- Rafiya Sultana
- School of Chemistry, University of Hyderabad, Hyderabad, 500046, India
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40
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Schifano JM, Cruz JW, Vvedenskaya IO, Edifor R, Ouyang M, Husson RN, Nickels BE, Woychik NA. tRNA is a new target for cleavage by a MazF toxin. Nucleic Acids Res 2016; 44:1256-70. [PMID: 26740583 PMCID: PMC4756823 DOI: 10.1093/nar/gkv1370] [Citation(s) in RCA: 74] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Accepted: 11/25/2015] [Indexed: 01/08/2023] Open
Abstract
Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA(Pro14) D-loop or within the tRNA(Lys43) anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth.
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Affiliation(s)
- Jason M Schifano
- Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ, USA
| | - Jonathan W Cruz
- Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ, USA
| | - Irina O Vvedenskaya
- Waksman Institute, Rutgers University, Piscataway, NJ, USA Department of Genetics, Rutgers University, Piscataway, NJ, USA
| | - Regina Edifor
- Division of Infectious Diseases, Boston Children's Hospital/Harvard Medical School, Boston, MA, USA
| | - Ming Ouyang
- Department of Computer Science, University of Massachusetts Boston, Boston, MA, USA
| | - Robert N Husson
- Division of Infectious Diseases, Boston Children's Hospital/Harvard Medical School, Boston, MA, USA
| | - Bryce E Nickels
- Waksman Institute, Rutgers University, Piscataway, NJ, USA Department of Genetics, Rutgers University, Piscataway, NJ, USA Member, Rutgers Cancer Institute of New Jersey, NJ, USA
| | - Nancy A Woychik
- Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ, USA Member, Rutgers Cancer Institute of New Jersey, NJ, USA
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41
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RD-1 encoded EspJ protein gets phosphorylated prior to affect the growth and intracellular survival of mycobacteria. Sci Rep 2015; 5:12717. [PMID: 26228622 PMCID: PMC4521147 DOI: 10.1038/srep12717] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2014] [Accepted: 07/02/2015] [Indexed: 01/12/2023] Open
Abstract
Mycobacterium tuberculosis (MTB) synchronizes a number of processes and controls a series of events to subvert host defense mechanisms for the sake of residing inside macrophages. Besides these, MTB also possesses a wide range of signal enzyme systems, including eleven serine threonine protein kinases (STPKs). The present study describes STPK modulated modification in one of the hypothetical proteins of the RD1 region; EspJ (ESX-1 secretion associated protein), which is predicted to be involved in virulence of MTB. We have employed knock-out MTB, and M. bovis BCG as a surrogate strain to elaborate the consequence of the phosphorylation of EspJ. The molecular and mass spectrometric analyses in this study, confirmed EspJ as one of the substrates of STPKs. The ectopic expression of phosphoablative mutants of espJ in M. bovis BCG also articulated the effect of phosphorylation on the growth and in survival of mycobacteria. Importantly, the level of phosphorylation of EspJ also differed between pathogenic H37 Rv (Rv) and non pathogenic H37 Ra (Ra) strains of MTB. This further suggested that to a certain extent, the STPKs mediated phosphorylation may be accountable, in determining the growth and in intra-cellular survival of mycobacteria.
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42
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Ahmed A, Das A, Mukhopadhyay S. Immunoregulatory functions and expression patterns of PE/PPE family members: Roles in pathogenicity and impact on anti-tuberculosis vaccine and drug design. IUBMB Life 2015; 67:414-27. [DOI: 10.1002/iub.1387] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2015] [Accepted: 04/29/2015] [Indexed: 01/27/2023]
Affiliation(s)
- Asma Ahmed
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD); Hyderabad, Telengana India
| | - Arghya Das
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD); Hyderabad, Telengana India
- Manipal University; Manipal Karnataka India
| | - Sangita Mukhopadhyay
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD); Hyderabad, Telengana India
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43
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Deng W, Zeng J, Xiang X, Li P, Xie J. PE11 (Rv1169c) selectively alters fatty acid components of Mycobacterium smegmatis and host cell interleukin-6 level accompanied with cell death. Front Microbiol 2015; 6:613. [PMID: 26157429 PMCID: PMC4477156 DOI: 10.3389/fmicb.2015.00613] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2015] [Accepted: 06/02/2015] [Indexed: 11/26/2022] Open
Abstract
PE/PPE family proteins, named after their conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains of N-terminal, are most intriguing aspects of pathologic mycobacterial genome. The roles of most members of this family remain unknown, although selected genes of this family are related to the virulence of Mycobacterium tuberculosis. In order to decipher the role of Rv1169c, the Mycobacterium smegmatis strain heterologous expressed this ORF was constructed and identified that Rv1169c was a cell wall associated protein with a novel function in modifying the cell wall fatty acids. The growth of Rv1169c expressing strain was affected under surface stress, acidic condition and antibiotics treatment. M. smegmatis expressing Rv1169c induced necrotic cell death of macrophage after infection and significantly decreased interlukin-6 production compared to controls. In general, these results underscore a proposing role of Rv1169c in virulence of M. tuberculosis, as it's role in the susceptibility of anti-mycobacteria factors caused by modified cell wall fatty acid, and the induced necrotic cell death by Rv1169c is crucial for M. tuberculosis virulence during infection.
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Affiliation(s)
- Wanyan Deng
- 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, China
| | - Jie Zeng
- 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, China
| | - Xiaohong Xiang
- 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, China
| | - Ping Li
- 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, 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, China
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Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 2014; 9:e112963. [PMID: 25409509 PMCID: PMC4237348 DOI: 10.1371/journal.pone.0112963] [Citation(s) in RCA: 6017] [Impact Index Per Article: 547.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Accepted: 10/16/2014] [Indexed: 02/06/2023] Open
Abstract
Advances in modern sequencing technologies allow us to generate sufficient data to analyze hundreds of bacterial genomes from a single machine in a single day. This potential for sequencing massive numbers of genomes calls for fully automated methods to produce high-quality assemblies and variant calls. We introduce Pilon, a fully automated, all-in-one tool for correcting draft assemblies and calling sequence variants of multiple sizes, including very large insertions and deletions. Pilon works with many types of sequence data, but is particularly strong when supplied with paired end data from two Illumina libraries with small e.g., 180 bp and large e.g., 3–5 Kb inserts. Pilon significantly improves draft genome assemblies by correcting bases, fixing mis-assemblies and filling gaps. For both haploid and diploid genomes, Pilon produces more contiguous genomes with fewer errors, enabling identification of more biologically relevant genes. Furthermore, Pilon identifies small variants with high accuracy as compared to state-of-the-art tools and is unique in its ability to accurately identify large sequence variants including duplications and resolve large insertions. Pilon is being used to improve the assemblies of thousands of new genomes and to identify variants from thousands of clinically relevant bacterial strains. Pilon is freely available as open source software.
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Affiliation(s)
- Bruce J. Walker
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
- * E-mail: (BJW); (AME)
| | - Thomas Abeel
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
- VIB Department of Plant Systems Biology, Ghent University, Ghent, Belgium
| | - Terrance Shea
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Margaret Priest
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Amr Abouelliel
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Sharadha Sakthikumar
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Christina A. Cuomo
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Qiandong Zeng
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Jennifer Wortman
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Sarah K. Young
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Ashlee M. Earl
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
- * E-mail: (BJW); (AME)
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45
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Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis. mBio 2014; 5:e02020. [PMID: 25370496 PMCID: PMC4222108 DOI: 10.1128/mbio.02020-14] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size—their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets.
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Tundup S, Mohareer K, Hasnain SE. Mycobacterium tuberculosis PE25/PPE41 protein complex induces necrosis in macrophages: Role in virulence and disease reactivation? FEBS Open Bio 2014; 4:822-8. [PMID: 25379378 PMCID: PMC4219985 DOI: 10.1016/j.fob.2014.09.001] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2014] [Revised: 08/28/2014] [Accepted: 09/06/2014] [Indexed: 02/03/2023] Open
Abstract
The Mycobacterium secreted protein PE25/PPE41 drives TNF-α secretion. PE25/PPE41 protein induces necrotic cell death, but not apoptosis, in macrophages. Necotic cell death induced by PE25/PPE41 is independent of TNF-α/NFκB/AP-1 pathways. PE25/PPE41 possibly acts as virulence factor, by an ‘immune quorum sensing’ mechanism. Necrotic cell death may help in mycobacterial dissemination and re-activation. Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence. The underlying mechanisms and the bacterial factors involved therein are not well understood. The Mycobacterium tuberculosis (M. tuberculosis) co-operonic PE25/PPE41 protein complex, similar to ESAT-6/CFP-10, belonging to the PE/PPE and ESAT-6 families of genes has co-expanded and co-evolved in the genomes of pathogenic mycobacteria. We report a novel role of this highly immunogenic PE25/PPE41 protein complex in inducing necrosis, but not apoptosis, in macrophages. We propose that these protein complexes of M. tuberculosis, secreted by similar/unique transport system (Type VII), have an important role in M. tuberculosis virulence and disease reactivation.
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Affiliation(s)
- Smanla Tundup
- Department of Microbiology, University of Chicago, Chicago, IL, 60637, USA
| | - Krishnaveni Mohareer
- Department of Biochemistry, School of Life Sciences, University of Hyderabad, Prof CR Rao Road, Hyderabad 500 046, India
| | - Seyed E Hasnain
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India ; Dr Reddy's Institute of Life Sciences, University of Hyderabad Campus, Prof CR Rao Road, Hyderabad 500046, India
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Gene cooption in Mycobacteria and search for virulence attributes: Comparative proteomic analyses of Mycobacterium tuberculosis, Mycobacterium indicus pranii and other mycobacteria. Int J Med Microbiol 2014; 304:742-8. [DOI: 10.1016/j.ijmm.2014.05.006] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2013] [Revised: 05/16/2014] [Accepted: 05/21/2014] [Indexed: 02/07/2023] Open
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Liu B, Zhang X, Huang H, Zhang Y, Zhou F, Wang G. A novel molecular typing method of Mycobacteria based on DNA barcoding visualization. J Clin Bioinforma 2014; 4:4. [PMID: 24555538 PMCID: PMC3931916 DOI: 10.1186/2043-9113-4-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Accepted: 02/10/2014] [Indexed: 11/10/2022] Open
Abstract
Different subtypes of Mycobacterium tuberculosis (MTB) may induce diverse severe human infections, and some of their symptoms are similar to other pathogenes, e.g. Nontuberculosis mycobacteria (NTM). So determination of mycobacterium subtypes facilitates the effective control of MTB infection and proliferation. This study exploits a novel DNA barcoding visualization method for molecular typing of 17 mycobacteria genomes published in the NCBI prokaryotic genome database. Three mycobacterium genes (Rv0279c, Rv3508 and Rv3514) from the PE/PPE family of MT Band were detected to best represent the inter-strain pathogenetic variations. An accurate and fast MTB substrain typing method was proposed based on the combination of the aforementioned three biomarker genes and the 16S rRNA gene. The protocol of establishing a bacterial substrain typing system used in this study may also be applied to the other pathogenes.
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Affiliation(s)
| | | | | | | | - Fengfeng Zhou
- Department of Pathogenobiology, Basic Medical College of Jilin University, Changchun, Jilin, China.
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Ang KC, Ibrahim P, Gam LH. Analysis of differentially expressed proteins in late-stationary growth phase of Mycobacterium tuberculosis H37Rv. Biotechnol Appl Biochem 2013; 61:153-64. [PMID: 23826872 DOI: 10.1002/bab.1137] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 06/23/2013] [Indexed: 11/09/2022]
Abstract
Mycobacterium tuberculosis is a causative agent of tuberculosis (TB). The ability of M. tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of M. tuberculosis H37Rv over three growth phases, namely mid-log (14-day culture), early stationary (28-day culture), and late stationary (50-day culture), was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry were used to generate proteome maps of M. tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared with the other two phases. These proteins were SucD, TrpD, and Rv2161c, which belong to metabolic pathway proteins; FadE5, AccD5, DesA1, and Rv1139c are proteins involved in cell wall or lipid biosynthesis, whereas TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1, was not detectable in the late-stationary phase, although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection.
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Affiliation(s)
- Kai-Cheen Ang
- School of Pharmaceutical Sciences, University Sains Malaysia, Minden, Penang, Malaysia
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Misra N, Panda PK. In search of actionable targets for agrigenomics and microalgal biofuel production: sequence-structural diversity studies on algal and higher plants with a focus on GPAT protein. OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY 2013; 17:173-86. [PMID: 23496307 DOI: 10.1089/omi.2012.0094] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The triacylglycerol (TAG) pathway provides several targets for genetic engineering to optimize microalgal lipid productivity. GPAT (glycerol-3-phosphate acyltransferase) is a crucial enzyme that catalyzes the initial step of TAG biosynthesis. Despite many recent biochemical studies, a comprehensive sequence-structure analysis of GPAT across diverse lipid-yielding organisms is lacking. Hence, we performed a comparative genomic analysis of plastid-located GPAT proteins from 7 microalgae and 3 higher plants species. The close evolutionary relationship observed between red algae/diatoms and green algae/plant lineages in the phylogenetic tree were further corroborated by motif and gene structure analysis. The predicted molecular weight, amino acid composition, Instability Index, and hydropathicity profile gave an overall representation of the biochemical features of GPAT protein across the species under study. Furthermore, homology models of GPAT from Chlamydomonas reinhardtii, Arabidopsis thaliana, and Glycine max provided deep insights into the protein architecture and substrate binding sites. Despite low sequence identity found between algal and plant GPATs, the developed models exhibited strikingly conserved topology consisting of 14α helices and 9β sheets arranged in two domains. However, subtle variations in amino acids of fatty acyl binding site were identified that might influence the substrate selectivity of GPAT. Together, the results will provide useful resources to understand the functional and evolutionary relationship of GPAT and potentially benefit in development of engineered enzyme for augmenting algal biofuel production.
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Affiliation(s)
- Namrata Misra
- Bioresources Engineering Department, CSIR-Institute of Minerals and Materials Technology, Bhubaneswar 751 013, Odisha, India
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