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Matys J, Turska-Szewczuk A, Gieroba B, Kurzylewska M, Pękala-Safińska A, Sroka-Bartnicka A. Evaluation of Proteomic and Lipidomic Changes in Aeromonas-Infected Trout Kidney Tissue with the Use of FT-IR Spectroscopy and MALDI Mass Spectrometry Imaging. Int J Mol Sci 2022; 23:ijms232012551. [PMID: 36293421 PMCID: PMC9604335 DOI: 10.3390/ijms232012551] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Revised: 10/13/2022] [Accepted: 10/15/2022] [Indexed: 11/16/2022] Open
Abstract
Aeromonas species are opportunistic bacteria causing a vast spectrum of human diseases, including skin and soft tissue infections, meningitis, endocarditis, peritonitis, gastroenteritis, and finally hemorrhagic septicemia. The aim of our research was to indicate the molecular alterations in proteins and lipids profiles resulting from Aeromonas sobria and A. salmonicida subsp. salmonicida infection in trout kidney tissue samples. We successfully applied FT-IR (Fourier transform infrared) spectroscopy and MALDI-MSI (matrix-assisted laser desorption/ionization mass spectrometry imaging) to monitor changes in the structure and compositions of lipids, secondary conformation of proteins, and provide useful information concerning disease progression. Our findings indicate that the following spectral bands’ absorbance ratios (spectral biomarkers) can be used to discriminate healthy tissue from pathologically altered tissue, for example, lipids (CH2/CH3), amide I/amide II, amide I/CH2 and amide I/CH3. Spectral data obtained from 10 single measurements of each specimen indicate numerous abnormalities concerning proteins, lipids, and phospholipids induced by Aeromonas infection, suggesting significant disruption of the cell membranes. Moreover, the increase in the content of lysolipids such as lysophosphosphatidylcholine was observed. The results of this study suggest the application of both methods MALDI-MSI and FT-IR as accurate methods for profiling biomolecules and identifying biochemical changes in kidney tissue during the progression of Aeromonas infection.
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Affiliation(s)
- Joanna Matys
- Department of Biopharmacy, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland
- Correspondence: (J.M.); (A.S.-B.)
| | - Anna Turska-Szewczuk
- Department of Genetics and Microbiology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
| | - Barbara Gieroba
- Independent Unit of Spectroscopy and Chemical Imaging, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland
| | - Maria Kurzylewska
- Department of Genetics and Microbiology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
| | - Agnieszka Pękala-Safińska
- Department of Preclinical Sciences and Infectious Diseases, Poznan University of Life Sciences, Wołyńska 35, 60-637 Poznań, Poland
| | - Anna Sroka-Bartnicka
- Department of Genetics and Microbiology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
- Independent Unit of Spectroscopy and Chemical Imaging, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland
- Correspondence: (J.M.); (A.S.-B.)
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Zhao T, Yang B, Li H, Qian A, Cong W, Sun W, Kang Y. Essential role of ascO for virulence of Aeromonas veronii and inducing apoptosis. JOURNAL OF FISH DISEASES 2022; 45:1477-1489. [PMID: 35749548 DOI: 10.1111/jfd.13676] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Revised: 06/06/2022] [Accepted: 06/10/2022] [Indexed: 06/15/2023]
Abstract
Aeromonas veronii is a significant pathogen that is capable of infecting humans, animals, and aquatic animals. The type III secretion system (T3SS) is intimately associated with bacterial pathogenicity. The ascO gene is an important core component of T3SS in A. veronii, but its function is still unclear. The ascO gene of A. veronii TH0426 was deleted by using the pRE112 suicide plasmid to study its function. The study results showed that the ability of ∆ascO to adhere and invade EPC cells was significantly reduced by 1.28 times. The toxicity of the mutant strain ΔascO to EPC cells was consistently significantly lower than wild-type strain TH0426 at 1, 2, and 4 h. The LD50 values of ∆ascO against zebrafish and Carassius auratus (C. auratus) were 53 and 15 times that of the wild-type strain. In addition, the bacterial load of the mutant strain ΔascO in blood, heart, liver, and spleen was lower than wild-type strain TH0426. The Hoechst staining showed that the apoptotic degree of EPC cells induced by the mutant strain ΔascO was lower than that of the wild-type strain TH0426. Furthermore, real-time quantitative PCR (RT-qPCR) analysis revealed lower expression levels of pro-apoptotic genes (including cytC, cas3, cas9, TNF-α, and IL-1β) in C. auratus tissues infected with the mutant strain ΔascO compared to the wild-type strain TH0426. The results of in vivo and in vitro experiments have shown that ascO gene mutation can reduce the adhesion and toxicity of A. veronii to EPC and reduce the level of apoptosis induced by A. veronii. As a result, these insights will help further elucidate the function of the ascO gene and thus contribute to understanding the pathogenesis of A. veronii.
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Affiliation(s)
- Tong Zhao
- College of Veterinary Medicine/College of Animal Science and Technology, Jilin Agricultural University, Changchun, China
- Marine College, Shandong University, Weihai, China
| | - Bintong Yang
- Marine College, Shandong University, Weihai, China
| | - Hongjin Li
- College of Veterinary Medicine/College of Animal Science and Technology, Jilin Agricultural University, Changchun, China
- Marine College, Shandong University, Weihai, China
| | - Aidong Qian
- College of Veterinary Medicine/College of Animal Science and Technology, Jilin Agricultural University, Changchun, China
| | - Wei Cong
- Marine College, Shandong University, Weihai, China
| | - Wuwen Sun
- College of Veterinary Medicine/College of Animal Science and Technology, Jilin Agricultural University, Changchun, China
| | - Yuanhuan Kang
- College of Veterinary Medicine/College of Animal Science and Technology, Jilin Agricultural University, Changchun, China
- Marine College, Shandong University, Weihai, China
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Stévenin V, Neefjes J. Control of host PTMs by intracellular bacteria: An opportunity toward novel anti-infective agents. Cell Chem Biol 2022; 29:741-756. [PMID: 35512694 DOI: 10.1016/j.chembiol.2022.04.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 03/15/2022] [Accepted: 04/15/2022] [Indexed: 02/08/2023]
Abstract
Intracellular bacteria have developed a multitude of mechanisms to influence the post-translational modifications (PTMs) of host proteins to pathogen advantages. The recent explosion of insights into the diversity and sophistication of host PTMs and their manipulation by infectious agents challenges us to formulate a comprehensive vision of this complex and dynamic facet of the host-pathogen interaction landscape. As new discoveries continue to shed light on the central roles of PTMs in infectious diseases, technological advances foster our capacity to detect old and new PTMs and investigate their control and impact during pathogenesis, opening new possibilities for chemical intervention and infection treatment. Here, we present a comprehensive overview of these pathogenic mechanisms and offer perspectives on how these insights may contribute to the development of a new class of therapeutics that are urgently needed to face rising antibiotic resistances.
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Affiliation(s)
- Virginie Stévenin
- Department of Cell and Chemical Biology, Oncode Institute, Leiden University Medical Center (LUMC), Leiden 2333 ZC, the Netherlands.
| | - Jacques Neefjes
- Department of Cell and Chemical Biology, Oncode Institute, Leiden University Medical Center (LUMC), Leiden 2333 ZC, the Netherlands
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Fasciano AC, Dasanayake GS, Estes MK, Zachos NC, Breault DT, Isberg RR, Tan S, Mecsas J. Yersinia pseudotuberculosis YopE prevents uptake by M cells and instigates M cell extrusion in human ileal enteroid-derived monolayers. Gut Microbes 2022; 13:1988390. [PMID: 34793276 PMCID: PMC8604394 DOI: 10.1080/19490976.2021.1988390] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Many pathogens use M cells to access the underlying Peyer's patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb), and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyer's patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.
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Affiliation(s)
- Alyssa C. Fasciano
- Graduate Program in Immunology, Tufts Graduate School of Biomedical Sciences, Boston, USA
| | - Gaya S. Dasanayake
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, USA
| | - Mary K. Estes
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, USA
| | - Nicholas C. Zachos
- Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, USA
| | - David T. Breault
- Division of Endocrinology, Boston Children’s Hospital, Department of Pediatrics, Harvard Medical School, Boston, USA
| | - Ralph R. Isberg
- Graduate Program in Immunology, Tufts Graduate School of Biomedical Sciences, Boston, USA,Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, USA
| | - Shumin Tan
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, USA
| | - Joan Mecsas
- Graduate Program in Immunology, Tufts Graduate School of Biomedical Sciences, Boston, USA,Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, USA,CONTACT Joan Mecsas Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, USA
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Role of the Yersinia pseudotuberculosis Virulence Plasmid in Pathogen-Phagocyte Interactions in Mesenteric Lymph Nodes. EcoSal Plus 2021; 9:eESP00142021. [PMID: 34910573 DOI: 10.1128/ecosalplus.esp-0014-2021] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Yersinia pseudotuberculosis is an Enterobacteriaceae family member that is commonly transmitted by the fecal-oral route to cause infections. From the small intestine, Y. pseudotuberculosis can invade through Peyer's patches and lymph vessels to infect the mesenteric lymph nodes (MLNs). Infection of MLNs by Y. pseudotuberculosis results in the clinical presentation of mesenteric lymphadenitis. MLNs are important for immune responses to intestinal pathogens and microbiota in addition to their clinical relevance to Y. pseudotuberculosis infections. A characteristic of Y. pseudotuberculosis infection in MLNs is the formation of pyogranulomas. Pyogranulomas are composed of neutrophils, inflammatory monocytes, and lymphocytes surrounding extracellular microcolonies of Y. pseudotuberculosis. Key elements of the complex pathogen-host interaction in MLNs have been identified using mouse infection models. Y. pseudotuberculosis requires the virulence plasmid pYV to induce the formation of pyogranulomas in MLNs. The YadA adhesin and the Ysc-Yop type III secretion system (T3SS) are encoded on pYV. YadA mediates bacterial binding to host receptors, which engages the T3SS to preferentially translocate seven Yop effectors into phagocytes. The effectors promote pathogenesis by blocking innate immune defenses such as superoxide production, degranulation, and inflammasome activation, resulting in survival and growth of Y. pseudotuberculosis. On the other hand, certain effectors can trigger immune defenses in phagocytes. For example, YopJ triggers activation of caspase-8 and an apoptotic cell death response in monocytes within pyogranulomas that limits dissemination of Y. pseudotuberculosis from MLNs to the bloodstream. YopE can be processed as an antigen by phagocytes in MLNs, resulting in T and B cell responses to Y. pseudotuberculosis. Immune responses to Y. pseudotuberculosis in MLNs can also be detrimental to the host in the form of chronic lymphadenopathy. This review focuses on interactions between Y. pseudotuberculosis and phagocytes mediated by pYV that concurrently promote pathogenesis and host defense in MLNs. We propose that MLN pyogranulomas are immunological arenas in which opposing pYV-driven forces determine the outcome of infection in favor of the pathogen or host.
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Wang P, Li J, He TT, Li N, Mo ZL, Nie P, Xie HX. Pathogenic characterization of Aeromonas salmonicida subsp. masoucida turbot isolate from China. JOURNAL OF FISH DISEASES 2020; 43:1145-1154. [PMID: 32720397 DOI: 10.1111/jfd.13224] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Revised: 07/01/2020] [Accepted: 07/02/2020] [Indexed: 06/11/2023]
Abstract
Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A. salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A. salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A. salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.
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Affiliation(s)
- Ping Wang
- School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao, China
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
| | - Jie Li
- Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China
| | - Tian Tian He
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
| | - Nan Li
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
| | - Zhao Lan Mo
- Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China
| | - Pin Nie
- School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao, China
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
- Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China
| | - Hai Xia Xie
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
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Yersinia pseudotuberculosis YopH targets SKAP2-dependent and independent signaling pathways to block neutrophil antimicrobial mechanisms during infection. PLoS Pathog 2020; 16:e1008576. [PMID: 32392230 PMCID: PMC7241846 DOI: 10.1371/journal.ppat.1008576] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Revised: 05/21/2020] [Accepted: 04/23/2020] [Indexed: 02/06/2023] Open
Abstract
Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6–7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation. Pathogenic Yersinia species carry a virulence plasmid encoding a type 3 secretion system that translocates 6–7 effector Yops into host cells. We demonstrate that YopH protects Yersinia pseudotuberculosis from neutrophil-produced reactive oxygen species (ROS) and degranulation by interfering with signaling pathways downstream of three major receptor classes in neutrophils. We show that a previously identified target of YopH, SKAP2, controls some of the pathways essential for YopH to inactivate during infection. SKAP2 is essential in mediating ROS production downstream of two major receptors; however, it is dispensable for degranulation from the three major receptors tested. Our study illustrates that YopH protects Y. pseudotuberculosis by blocking both SKAP2-dependent and independent signaling pathways that regulate several neutrophil functions.
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Redundant and Cooperative Roles for Yersinia pestis Yop Effectors in the Inhibition of Human Neutrophil Exocytic Responses Revealed by Gain-of-Function Approach. Infect Immun 2020; 88:IAI.00909-19. [PMID: 31871100 DOI: 10.1128/iai.00909-19] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 12/16/2019] [Indexed: 12/13/2022] Open
Abstract
Yersinia pestis causes a rapid, lethal disease referred to as plague. Y. pestis actively inhibits the innate immune system to generate a noninflammatory environment during early stages of infection to promote colonization. The ability of Y. pestis to create this early noninflammatory environment is in part due to the action of seven Yop effector proteins that are directly injected into host cells via a type 3 secretion system (T3SS). While each Yop effector interacts with specific host proteins to inhibit their function, several Yop effectors either target the same host protein or inhibit converging signaling pathways, leading to functional redundancy. Previous work established that Y. pestis uses the T3SS to inhibit neutrophil respiratory burst, phagocytosis, and release of inflammatory cytokines. Here, we show that Y. pestis also inhibits release of granules in a T3SS-dependent manner. Moreover, using a gain-of-function approach, we discovered previously hidden contributions of YpkA and YopJ to inhibition and that cooperative actions by multiple Yop effectors are required to effectively inhibit degranulation. Independent from degranulation, we also show that multiple Yop effectors can inhibit synthesis of leukotriene B4 (LTB4), a potent lipid mediator released by neutrophils early during infection to promote inflammation. Together, inhibition of these two arms of the neutrophil response likely contributes to the noninflammatory environment needed for Y. pestis colonization and proliferation.
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Uribe-Querol E, Rosales C. Control of Phagocytosis by Microbial Pathogens. Front Immunol 2017; 8:1368. [PMID: 29114249 PMCID: PMC5660709 DOI: 10.3389/fimmu.2017.01368] [Citation(s) in RCA: 159] [Impact Index Per Article: 19.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2017] [Accepted: 10/05/2017] [Indexed: 12/17/2022] Open
Abstract
Phagocytosis is a fundamental process of cells to capture and ingest foreign particles. Small unicellular organisms such as free-living amoeba use this process to acquire food. In pluricellular organisms, phagocytosis is a universal phenomenon that all cells are able to perform (including epithelial, endothelial, fibroblasts, etc.), but some specialized cells (such as neutrophils and macrophages) perform this very efficiently and were therefore named professional phagocytes by Rabinovitch. Cells use phagocytosis to capture and clear all particles larger than 0.5 µm, including pathogenic microorganisms and cellular debris. Phagocytosis involves a series of steps from recognition of the target particle, ingestion of it in a phagosome (phagocytic vacuole), maturation of this phagosome into a phagolysosome, to the final destruction of the ingested particle in the robust antimicrobial environment of the phagolysosome. For the most part, phagocytosis is an efficient process that eliminates invading pathogens and helps maintaining homeostasis. However, several pathogens have also evolved different strategies to prevent phagocytosis from proceeding in a normal way. These pathogens have a clear advantage to perpetuate the infection and continue their replication. Here, we present an overview of the phagocytic process with emphasis on the antimicrobial elements professional phagocytes use. We also summarize the current knowledge on the microbial strategies different pathogens use to prevent phagocytosis either at the level of ingestion, phagosome formation, and maturation, and even complete escape from phagosomes.
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Affiliation(s)
- Eileen Uribe-Querol
- División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, Mexico City, Mexico
| | - Carlos Rosales
- Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
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Lee WL, Singaravelu P, Wee S, Xue B, Ang KC, Gunaratne J, Grimes JM, Swaminathan K, Robinson RC. Mechanisms of Yersinia YopO kinase substrate specificity. Sci Rep 2017; 7:39998. [PMID: 28051168 PMCID: PMC5209680 DOI: 10.1038/srep39998] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2016] [Accepted: 11/30/2016] [Indexed: 02/06/2023] Open
Abstract
Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO's substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.
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Affiliation(s)
- Wei Lin Lee
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
| | - Pavithra Singaravelu
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
- Department of Biological Sciences, National University of Singapore, Singapore
| | - Sheena Wee
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
| | - Bo Xue
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
| | - Khay Chun Ang
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
| | - Jayantha Gunaratne
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
- Department of Anatomy, National University of Singapore, Singapore
| | - Jonathan M. Grimes
- Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, UK
- Diamond Light Source Ltd., UK
| | | | - Robert C. Robinson
- Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
- Department of Biochemistry, National University of Singapore, Singapore
- NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, 636921, Singapore
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore
- Lee Kong Chan School of Medicine, 50 Nanyang Avenue, 639798, Singapore
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11
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Origgi FC, Benedicenti O, Segner H, Sattler U, Wahli T, Frey J. Aeromonas salmonicida type III secretion system-effectors-mediated immune suppression in rainbow trout (Oncorhynchus mykiss). FISH & SHELLFISH IMMUNOLOGY 2017; 60:334-345. [PMID: 27923746 DOI: 10.1016/j.fsi.2016.12.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Revised: 11/28/2016] [Accepted: 12/02/2016] [Indexed: 06/06/2023]
Abstract
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen in aquaculture. Together with other pathogens, it is characterized by the presence of a type 3 secretion system (T3SS). The T3SS is the main virulence mechanism of A. salmonicida. It is used by the bacterium to secrete and translocate several toxins and effector proteins into the host cell. Some of these factors have a detrimental impact on the integrity of the cell cytoskeleton, likely contributing to impair phagocytosis. Furthermore, it has been suggested that effectors of the T3SS are able to modulate the host's immune response. Here we present the first partial characterization of the immune response in rainbow trout (Oncorhynchus mykiss) infected with distinct strains of A. salmonicida either carrying (i) a fully functional T3SS or (ii) a functionally impaired T3SS or (iii) devoid of T3SS ("cured" strain). Infection with an A. salmonicida strain either carrying a fully functional or a secretion-impaired T3SS was associated with a strong and persistent immune suppression. However, the infection appeared to be fatal only in the presence of a fully functional T3SS. In contrast, the absence of T3SS was neither associated with immune suppression nor fish death. These findings suggest that the T3SS and T3SS-delivered effector molecules and toxins of A. salmonicida do not only impair the host cells' cytoskeleton thus damaging cell physiology and phagocytosis, but also heavily affect the transcription of critical immune mediators including the shut-down of important warning signals to recognize infection and induce immune defense.
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Affiliation(s)
- F C Origgi
- Institute of Veterinary Bacteriology, University of Bern, Bern-CH, Switzerland; Centre for Fish and Wildlife Health (FIWI), University of Bern, Bern-CH, Switzerland.
| | - O Benedicenti
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, UK
| | - H Segner
- Centre for Fish and Wildlife Health (FIWI), University of Bern, Bern-CH, Switzerland
| | - U Sattler
- Centre for Fish and Wildlife Health (FIWI), University of Bern, Bern-CH, Switzerland
| | - T Wahli
- Centre for Fish and Wildlife Health (FIWI), University of Bern, Bern-CH, Switzerland
| | - J Frey
- Institute of Veterinary Bacteriology, University of Bern, Bern-CH, Switzerland
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12
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13
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Zhang L, Mei M, Yu C, Shen W, Ma L, He J, Yi L. The Functions of Effector Proteins in Yersinia Virulence. Pol J Microbiol 2016; 65:5-12. [PMID: 27281989 DOI: 10.5604/17331331.1197324] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Yersinia species are bacterial pathogens that can cause plague and intestinal diseases after invading into human cells through the Three Secretion System (TTSS). The effect of pathogenesis is mediated by Yersinia outer proteins (Yop) and manifested as down-regulation of the cytokine genes expression by inhibiting nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. In addition, its pathogenesis can also manipulate the disorder of host innate immune system and cell death such as apoptosis, pyroptosis, and autophagy. Among the Yersinia effector proteins, YopB and YopD assist the injection of other virulence effectors into the host cytoplasm, while YopE, YopH, YopJ, YopO, and YopT target on disrupting host cell signaling pathways in the host cytosols. Many efforts have been applied to reveal that intracellular proteins such as Rho-GTPase, and transmembrane receptors such as Toll-like receptors (TLRs) both play critical roles in Yersinia pathogenesis, establishing a connection between the pathogenic process and the signaling response. This review will mainly focus on how the effector proteins of Yersinia modulate the intrinsic signals in host cells and disturb the innate immunity of hosts through TTSS.
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14
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Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia. Semin Cell Dev Biol 2016; 60:155-167. [PMID: 27448494 PMCID: PMC7082150 DOI: 10.1016/j.semcdb.2016.07.019] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2016] [Revised: 07/15/2016] [Accepted: 07/18/2016] [Indexed: 01/07/2023]
Abstract
Bacterial pathogens display an impressive arsenal of molecular mechanisms that allow survival in diverse host niches. Subversion of plasma membrane and cytoskeletal functions are common themes associated to infection by both extracellular and intracellular pathogens. Moreover, intracellular pathogens modify the structure/stability of their membrane-bound compartments and escape degradation from phagocytic or autophagic pathways. Here, we review the manipulation of host membranes by Listeria monocytogenes, Francisella tularensis, Shigella flexneri and Yersinia spp. These four bacterial model pathogens exemplify generalized strategies as well as specific features observed during bacterial infection processes.
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15
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Menanteau-Ledouble S, Kumar G, Saleh M, El-Matbouli M. Aeromonas salmonicida: updates on an old acquaintance. DISEASES OF AQUATIC ORGANISMS 2016; 120:49-68. [PMID: 27304870 DOI: 10.3354/dao03006] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Aeromonas salmonicida is the oldest known infectious agent to be linked to fish disease and constitutes a major bacterial pathogen of fish, in particular of salmonids. This bacterium can be found almost worldwide in both marine and freshwater environments and has been divided into several sub-species. In this review, we present the most recent developments concerning our understanding of this pathogen, including how the characterization of new isolates from non-salmonid hosts suggests a more nuanced picture of the importance of the so‑called 'atypical isolates'. We also describe the clinical presentation regarding the infection across several fish species and discuss what is known about the virulence of A. salmonicida and, in particular, the role that the type 3 secretion system might play in suppressing the immune response of its hosts. Finally, isolates have displayed varied levels of antibiotic resistance. Hence, we review a number of solutions that have been developed both to prevent outbreaks and to treat them once they occur, including the application of pre- and probiotic supplements.
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Affiliation(s)
- Simon Menanteau-Ledouble
- Clinical Division of Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria
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16
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Abstract
The human pathogens
Yersinia pseudotuberculosis and
Yersinia enterocolitica cause enterocolitis, while
Yersinia pestis is responsible for pneumonic, bubonic, and septicaemic plague. All three share an infection strategy that relies on a virulence factor arsenal to enable them to enter, adhere to, and colonise the host while evading host defences to avoid untimely clearance. Their arsenal includes a number of adhesins that allow the invading pathogens to establish a foothold in the host and to adhere to specific tissues later during infection. When the host innate immune system has been activated, all three pathogens produce a structure analogous to a hypodermic needle. In conjunction with the translocon, which forms a pore in the host membrane, the channel that is formed enables the transfer of six ‘effector’ proteins into the host cell cytoplasm. These proteins mimic host cell proteins but are more efficient than their native counterparts at modifying the host cell cytoskeleton, triggering the host cell suicide response. Such a sophisticated arsenal ensures that yersiniae maintain the upper hand despite the best efforts of the host to counteract the infecting pathogen.
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Affiliation(s)
- Steve Atkinson
- Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, UK
| | - Paul Williams
- Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham, UK
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17
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Pha K, Navarro L. Yersinia type III effectors perturb host innate immune responses. World J Biol Chem 2016; 7:1-13. [PMID: 26981193 PMCID: PMC4768113 DOI: 10.4331/wjbc.v7.i1.1] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 09/02/2015] [Accepted: 11/04/2015] [Indexed: 02/05/2023] Open
Abstract
The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.
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18
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Alugubelly N, Hercik K, Kibler P, Nanduri B, Edelmann MJ. Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2016; 1864:562-9. [PMID: 26854600 DOI: 10.1016/j.bbapap.2016.02.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/05/2015] [Revised: 01/20/2016] [Accepted: 02/03/2016] [Indexed: 12/22/2022]
Abstract
UNLABELLED Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Y.enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (including Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (including Rab1 and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions. STATEMENT OF SIGNIFICANCE OF THE STUDY We describe a proteome of Yersinia enterocolitica-infected HeLa cells, including a description of specific proteins differentially expressed upon infection, molecular functions as well as pathways altered during infection. This proteomic study can lead to a better understanding of Y. enterocolitica pathogenesis in human epithelial cells.
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Affiliation(s)
- Navatha Alugubelly
- Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, USA
| | - Kamil Hercik
- Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, USA
| | - Peter Kibler
- Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA
| | - Bindu Nanduri
- Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, USA
| | - Mariola J Edelmann
- Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA.
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19
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Dominguez R. Subversive bacteria reveal new tricks in their cytoskeleton-hijacking arsenal. Nat Struct Mol Biol 2015; 22:178-9. [PMID: 25736086 DOI: 10.1038/nsmb.2976] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- Roberto Dominguez
- Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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20
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Abstract
Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review.
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Affiliation(s)
- Yingyu Mao
- a Department of Biological Sciences; Center for Cancer, Genetic Diseases, and Gene Regulation; Fordham University ; Bronx , NY , USA
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21
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Lee WL, Grimes JM, Robinson RC. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization. Nat Struct Mol Biol 2015; 22:248-55. [PMID: 25664724 DOI: 10.1038/nsmb.2964] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2014] [Accepted: 12/30/2014] [Indexed: 11/09/2022]
Abstract
Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.
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Affiliation(s)
- Wei Lin Lee
- 1] Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore. [2] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - Jonathan M Grimes
- 1] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. [2] Diamond Light Source, Oxfordshire, UK
| | - Robert C Robinson
- 1] Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore. [2] Department of Biochemistry, National University of Singapore, Singapore
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22
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Pha K, Wright ME, Barr TM, Eigenheer RA, Navarro L. Regulation of Yersinia protein kinase A (YpkA) kinase activity by multisite autophosphorylation and identification of an N-terminal substrate-binding domain in YpkA. J Biol Chem 2014; 289:26167-26177. [PMID: 25086045 DOI: 10.1074/jbc.m114.601153] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The serine/threonine protein kinase YpkA is an essential virulence factor produced by pathogenic Yersinia species. YpkA is delivered into host mammalian cells via a type III secretion system and localizes to the inner side of the plasma membrane. We have previously shown that YpkA binds to and phosphorylates the α subunit of the heterotrimeric G protein complex, Gαq, resulting in inhibition of Gαq signaling. To identify residues in YpkA involved in substrate binding activity we generated GFP-YpkA N-terminal deletion mutants and performed coimmunoprecipitation experiments. We located a substrate-binding domain on amino acids 40-49 of YpkA, which lies within the previously identified membrane localization domain on YpkA. Deletion of amino acids 40-49 on YpkA interfered with substrate binding, substrate phosphorylation and substrate inhibition. Autophosphorylation regulates the kinase activity of YpkA. To dissect the mechanism by which YpkA transmits signals, we performed nano liquid chromatography coupled to tandem mass spectrometry to map in vivo phosphorylation sites. Multiple serine phosphorylation sites were identified in the secretion/translocation region, kinase domain, and C-terminal region of YpkA. Using site-directed mutagenesis we generated multiple YpkA constructs harboring specific serine to alanine point mutations. Our results demonstrate that multiple autophosphorylation sites within the N terminus regulate YpkA kinase activation, whereas mutation of serine to alanine within the C terminus of YpkA had no effect on kinase activity. YpkA autophosphorylation on multiple sites may be a strategy used by pathogenic Yersinia to prevent inactivation of this important virulence protein by host proteins.
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Affiliation(s)
- Khavong Pha
- Department of Microbiology and Molecular Genetics, University of California, Davis, California 95616 and
| | - Matthew E Wright
- Department of Microbiology and Molecular Genetics, University of California, Davis, California 95616 and
| | - Tasha M Barr
- Department of Microbiology and Molecular Genetics, University of California, Davis, California 95616 and
| | - Richard A Eigenheer
- Proteomics Core Facility, Genome Center, University of California-Davis, Davis, California 95616
| | - Lorena Navarro
- Department of Microbiology and Molecular Genetics, University of California, Davis, California 95616 and.
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23
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Popoff MR. Bacterial factors exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host. Small GTPases 2014; 5:28209. [PMID: 25203748 DOI: 10.4161/sgtp.28209] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Actin cytoskeleton is a main target of many bacterial pathogens. Among the multiple regulation steps of the actin cytoskeleton, bacterial factors interact preferentially with RhoGTPases. Pathogens secrete either toxins which diffuse in the surrounding environment, or directly inject virulence factors into target cells. Bacterial toxins, which interfere with RhoGTPases, and to some extent with RasGTPases, catalyze a covalent modification (ADPribosylation, glucosylation, deamidation, adenylation, proteolysis) blocking these molecules in their active or inactive state, resulting in alteration of epithelial and/or endothelial barriers, which contributes to dissemination of bacteria in the host. Injected bacterial virulence factors preferentially manipulate the RhoGTPase signaling cascade by mimicry of eukaryotic regulatory proteins leading to local actin cytoskeleton rearrangement, which mediates bacterial entry into host cells or in contrast escape to phagocytosis and immune defense. Invasive bacteria can also manipulate RhoGTPase signaling through recognition and stimulation of cell surface receptor(s). Changes in RhoGTPase activation state is sensed by the innate immunity pathways and allows the host cell to adapt an appropriate defense response.
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Affiliation(s)
- Michel R Popoff
- Unité des Bactéries anaérobies et Toxines; Institut Pasteur; Paris, France
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24
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Ke Y, Chen Z, Yang R. Yersinia pestis: mechanisms of entry into and resistance to the host cell. Front Cell Infect Microbiol 2013; 3:106. [PMID: 24400226 PMCID: PMC3871965 DOI: 10.3389/fcimb.2013.00106] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2013] [Accepted: 12/10/2013] [Indexed: 12/28/2022] Open
Abstract
During infection, Yersinia, a facultative intracellular bacterial species, exhibits the ability to first invade host cells and then counteract phagocytosis by the host cells. During these two distinct stages, invasion or antiphagocytic factors assist bacteria in manipulating host cells to accomplish each of these functions; however, the mechanism through which Yersinia regulates these functions during each step remains unclear. Here, we discuss those factors that seem to function reversely and give some hypothesis about how bacteria switch between the two distinct status.
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Affiliation(s)
- Yuehua Ke
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China ; Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences Beijing, China
| | - Zeliang Chen
- Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China
| | - Ruifu Yang
- Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences Beijing, China
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25
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Yersinia enterocolitica inhibits Salmonella enterica serovar Typhimurium and Listeria monocytogenes cellular uptake. Infect Immun 2013; 82:174-83. [PMID: 24126528 DOI: 10.1128/iai.00984-13] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Yersinia enterocolitica biovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely, Salmonella enterica serovar Typhimurium uses a T3SS encoded by Salmonella pathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake of S. Typhimurium and Y. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other. Y. enterocolitica reduces S. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using an S. Typhimurium SPI1 mutant alone. However, Y. enterocolitica had no effect on S. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells. Y. enterocolitica was also able to inhibit the invasion of epithelial and macrophage-like cells by Listeria monocytogenes. Y. enterocolitica mutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blocking S. Typhimurium uptake by epithelial cells. S. Typhimurium encodes a LuxR homolog, SdiA, which detects N-acylhomoserine lactones (AHLs) produced by Y. enterocolitica and upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating the S. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed by Y. enterocolitica.
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26
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Vanden Bergh P, Frey J. Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system. Microb Biotechnol 2013; 7:381-400. [PMID: 24119189 PMCID: PMC4229320 DOI: 10.1111/1751-7915.12091] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2013] [Revised: 08/27/2013] [Accepted: 08/28/2013] [Indexed: 11/30/2022] Open
Abstract
Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.
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Affiliation(s)
- Philippe Vanden Bergh
- Institute of Veterinary Bacteriology, University of Bern, Länggassstrasse 122, Bern, Switzerland
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27
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Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors. Trends Microbiol 2013; 21:430-41. [DOI: 10.1016/j.tim.2013.06.008] [Citation(s) in RCA: 100] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2013] [Revised: 05/08/2013] [Accepted: 06/18/2013] [Indexed: 11/17/2022]
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28
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Wolters M, Boyle EC, Lardong K, Trülzsch K, Steffen A, Rottner K, Ruckdeschel K, Aepfelbacher M. Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein. J Biol Chem 2013; 288:23543-53. [PMID: 23803609 DOI: 10.1074/jbc.m112.448662] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.
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Affiliation(s)
- Manuel Wolters
- Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
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29
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Structural basis of eukaryotic cell targeting by type III secretion system (T3SS) effectors. Res Microbiol 2013; 164:605-19. [PMID: 23541478 DOI: 10.1016/j.resmic.2013.03.019] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Accepted: 02/27/2013] [Indexed: 02/06/2023]
Abstract
Type III secretion systems (T3SS) are macromolecular complexes that translocate a wide number of effector proteins into eukaryotic host cells. Once within the cytoplasm, many T3SS effectors mimic the structure and/or function of eukaryotic proteins in order to manipulate signaling cascades, and thus play pivotal roles in colonization, invasion, survival and virulence. Structural biology techniques have played key roles in the unraveling of bacterial strategies employed for mimicry and targeting. This review provides an overall view of our current understanding of structure and function of T3SS effectors, as well as of the different classes of eukaryotic proteins that are targeted and the consequences for the infected cell.
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30
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Rosales-Reyes R, Aubert DF, Tolman JS, Amer AO, Valvano MA. Burkholderia cenocepacia type VI secretion system mediates escape of type II secreted proteins into the cytoplasm of infected macrophages. PLoS One 2012; 7:e41726. [PMID: 22848580 PMCID: PMC3405007 DOI: 10.1371/journal.pone.0041726] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Accepted: 06/27/2012] [Indexed: 11/18/2022] Open
Abstract
Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.
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Affiliation(s)
- Roberto Rosales-Reyes
- Centre for Human Immunology, Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Laboratorio de Infectología, Microbiología e Inmunología Clínicas, Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, México
| | - Daniel F. Aubert
- Centre for Human Immunology, Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
| | - Jennifer S. Tolman
- Centre for Human Immunology, Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
| | - Amal O. Amer
- Centre for Microbial Interface Biology, Department of Microbial Infection and Immunity and the Department of Internal Medicine, Ohio State University, Columbus, Ohio, United States of America
| | - Miguel A. Valvano
- Centre for Human Immunology, Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- * E-mail:
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Wang XX, Sun RJ, Wu M, Li T, Zhang Y, Chen L. Differential protein expression in EC304 gastric cancer cells induced by alphastatin. Asian Pac J Cancer Prev 2012; 13:1667-74. [PMID: 22799386 DOI: 10.7314/apjcp.2012.13.4.1667] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
OBJECTIVE To explore the differential protein expression profile in EC304 gastric cancer cells induced by alphastatin. METHODS Cultured EC304 cells in the exponential phase of growth were randomly divided into alphastatin and control groups. Total proteins were extracted and the two dimensional electrophoresis (2-DE) technique was applied to analyze differences in expression with ImageMaster 2D Platinum 5.0 software. Proteins were identified using the MASCOT database and selected differently expressed proteins were characterised by western blotting and immunofluorescence. RESULTS 1350 ± 90 protein spots were detected by the ImageMaster software in the 2-DE gel images from the control and alphastatin groups. The match rate was about 72-80% for the spectrum profiles, with 29 significantly different protein spots being identified, 10 upregulated, 16 downregulated, two new and one lost. The MASCOT search scores were 64-666 and the peptide matching numbers were 3-27 with sequence coverage of 8-62%. Twenty-three proteins were checked by mass spectrometry, including decrease in Nm23 and profilin-2 isoform b associated with the regulation of actin multimerisation induced by extracellular signals. CONCLUSION The proteome in EC304 cells is dramatically altered by alphastatin, which appears to play an important role in modulating cellular activity and anti-angiogenesis by regulating protein expression and signal transduction pathways through Nm23 and profilin-2 isoform b, providing new research directions for anti-angiogenic therapy of gastric cancer.
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Affiliation(s)
- Xin-Xin Wang
- Department of General Surgery, Chinese People's Liberation Army General Hospital, Beijing, China
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Abstract
Protein phosphorylation on tyrosine has emerged as a key device in the control of numerous cellular functions in bacteria. In this article, we review the structure and function of bacterial tyrosine kinases and phosphatases. Phosphorylation is catalyzed by autophosphorylating adenosine triphosphate-dependent enzymes (bacterial tyrosine (BY) kinases) that are characterized by the presence of Walker motifs. The reverse reaction is catalyzed by three classes of enzymes: the eukaryotic-like phosphatases (PTPs) and dual-specific phosphatases; the low molecular weight protein-tyrosine phosphatases (LMW-PTPs); and the polymerase–histidinol phosphatases (PHP). Many BY kinases and tyrosine phosphatases can utilize host cell proteins as substrates, thereby contributing to bacterial pathogenicity. Bacterial tyrosine phosphorylation/dephosphorylation is also involved in biofilm formation and community development. The Porphyromonas gingivalis tyrosine phosphatase Ltp1 is involved in a restraint pathway that regulates heterotypic community development with Streptococcus gordonii. Ltp1 is upregulated by contact with S. gordonii and Ltp1 activity controls adhesin expression and levels of the interspecies signal AI-2.
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Sironi M, Guerini FR, Agliardi C, Biasin M, Cagliani R, Fumagalli M, Caputo D, Cassinotti A, Ardizzone S, Zanzottera M, Bolognesi E, Riva S, Kanari Y, Miyazawa M, Clerici M. An evolutionary analysis of RAC2 identifies haplotypes associated with human autoimmune diseases. Mol Biol Evol 2011; 28:3319-29. [PMID: 21680873 DOI: 10.1093/molbev/msr164] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2023] Open
Abstract
The human RAC2 gene encodes a small GTP-binding protein with a pivotal role in immune activation and in the induction of peripheral immune tolerance through restimulation-induced cell death (RICD). Different human pathogens target the protein product of RAC2, suggesting that the gene may be subject to natural selection, and that variants in RAC2 may affect immunological phenotypes in humans. We scanned the genomic region encompassing the entire transcription unit for the presence of putative noncoding regulatory elements conserved across mammals. This information was used to select two RAC2 gene regions and analyze their intraspecific genetic diversity. Results suggest that a region covering the 3' untranslated region has been a target of multiallelic balancing selection (or diversifying selection), and three major RAC2 haplogroups occur in human populations. Haplotypes belonging to one of these clades are associated with increased susceptibility to multiple sclerosis (P = 0.022) and earlier onset of disease symptoms (P = 0.025). This same haplogroup is significantly more common in patients with Crohn's disease compared with healthy controls (P = 0.048). These data reinforce recent evidences that susceptibility alleles/haplotypes are shared among multiple autoimmune disorders and support a causal "role for RAC2" variants in the pathogenesis of autoimmune diseases. Other genes with a role in RICD have previously been associated with autoimmunity in humans, suggesting that this pathway and RAC2 may represent novel therapeutic targets in autoimmune disorders.
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Affiliation(s)
- Manuela Sironi
- Bioinformatics Laboratory, Scientific Institute IRCCS E. Medea, Bosisio Parini (LC), Italy
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Rosales-Reyes R, Skeldon AM, Aubert DF, Valvano MA. The Type VI secretion system of Burkholderia cenocepacia affects multiple Rho family GTPases disrupting the actin cytoskeleton and the assembly of NADPH oxidase complex in macrophages. Cell Microbiol 2011; 14:255-73. [PMID: 22023353 DOI: 10.1111/j.1462-5822.2011.01716.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/16/2022]
Abstract
Burkholderia cenocepacia is a Gram-negative opportunistic pathogen of patients with cystic fibrosis and chronic granulomatous disease. The bacterium survives intracellularly in macrophages within a membrane-bound vacuole (BcCV) that precludes the fusion with lysosomes. The underlying cellular mechanisms and bacterial molecules mediating these phenotypes are unknown. Here, we show that intracellular B. cenocepacia expressing a type VI secretion system (T6SS) affects the activation of the Rac1 and Cdc42 RhoGTPase by reducing the cellular pool of GTP-bound Rac1 and Cdc42. The T6SS also increases the cellular pool of GTP-bound RhoA and decreases cofilin activity. These effects lead to abnormal actin polymerization causing collapse of lamellipodia and failure to retract the uropod. The T6SS also prevents the recruitment of soluble subunits of the NADPH oxidase complex including Rac1 to the BcCV membrane, but is not involved in the BcCV maturation arrest. Therefore, T6SS-mediated deregulation of Rho family GTPases is a common mechanism linking disruption of the actin cytoskeleton and delayed NADPH oxidase activation in macrophages infected with B. cenocepacia.
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Affiliation(s)
- Roberto Rosales-Reyes
- Centre for Human Immunology, Department of Microbiology and Immunology Department of Medicine, University of Western Ontario, London, ON N6A 5C1, Canada
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Schmidt G. Yersinia enterocolitica outer protein T (YopT). Eur J Cell Biol 2011; 90:955-8. [DOI: 10.1016/j.ejcb.2010.12.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2010] [Revised: 12/23/2010] [Accepted: 12/23/2010] [Indexed: 01/18/2023] Open
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Position effect of effectors. Nat Rev Microbiol 2010. [DOI: 10.1038/nrmicro2286] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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