|
1
|
Walch P, Broz P. Viral-bacterial co-infections screen in vitro reveals molecular processes affecting pathogen proliferation and host cell viability. Nat Commun 2024; 15:8595. [PMID: 39366977 PMCID: PMC11452664 DOI: 10.1038/s41467-024-52905-2] [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: 03/14/2024] [Accepted: 09/24/2024] [Indexed: 10/06/2024] Open
Abstract
The broadening of accessible methodologies has enabled mechanistic insights into single-pathogen infections, yet the molecular mechanisms underlying co-infections remain largely elusive, despite their clinical frequency and relevance, generally exacerbating symptom severity and fatality. Here, we describe an unbiased in vitro screening of pairwise co-infections in a murine macrophage model, quantifying pathogen proliferation and host cell death in parallel over time. The screen revealed that the majority of interactions are antagonistic for both metrics, highlighting general patterns depending on the pathogen virulence strategy. We subsequently decipher two distinct molecular interaction points: Firstly, murine Adenovirus 3 modifies ASC-dependent inflammasome responses in murine macrophages, altering host cell death and cytokine production, thereby impacting secondary Salmonella infection. Secondly, murine Adenovirus 2 infection triggers upregulation of Mprip, a crucial mediator of phagocytosis, which in turn causes increased Yersinia uptake, specifically in virus pre-infected bone-marrow-derived macrophages. This work therefore encompasses both a first-of-its-kind systematic assessment of host-pathogen-pathogen interactions, and mechanistic insight into molecular mediators during co-infection.
Collapse
Affiliation(s)
- Philipp Walch
- University of Lausanne, Department of Immunobiology, Chemin des Boveresses 155, CH-1066, Epalinges, Switzerland
| | - Petr Broz
- University of Lausanne, Department of Immunobiology, Chemin des Boveresses 155, CH-1066, Epalinges, Switzerland.
| |
Collapse
|
|
2
|
Fang X, Kang L, Qiu YF, Li ZS, Bai Y. Yersinia enterocolitica in Crohn’s disease. Front Cell Infect Microbiol 2023; 13:1129996. [PMID: 36968108 PMCID: PMC10031030 DOI: 10.3389/fcimb.2023.1129996] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Accepted: 02/13/2023] [Indexed: 03/11/2023] Open
Abstract
Increasing attention is being paid to the unique roles gut microbes play in both physiological and pathological processes. Crohn’s disease (CD) is a chronic, relapsing, inflammatory disease of the gastrointestinal tract with unknown etiology. Currently, gastrointestinal infection has been proposed as one initiating factor of CD. Yersinia enterocolitica, a zoonotic pathogen that exists widely in nature, is one of the most common bacteria causing acute infectious gastroenteritis, which displays clinical manifestations similar to CD. However, the specific role of Y. enterocolitica in CD is controversial. In this Review, we discuss the current knowledge on how Y. enterocolitica and derived microbial compounds may link to the pathogenesis of CD. We highlight examples of Y. enterocolitica-targeted interventions in the diagnosis and treatment of CD, and provide perspectives for future basic and translational investigations on this topic.
Collapse
Affiliation(s)
| | | | | | | | - Yu Bai
- *Correspondence: Zhao-Shen Li, ; Yu Bai,
| |
Collapse
|
|
3
|
Cao S, Jiao Y, Jiang W, Wu Y, Qin S, Ren Y, You Y, Tan Y, Guo X, Chen H, Zhang Y, Wu G, Wang T, Zhou Y, Song Y, Cui Y, Shao F, Yang R, Du Z. Subversion of GBP-mediated host defense by E3 ligases acquired during Yersinia pestis evolution. Nat Commun 2022; 13:4526. [PMID: 35927280 PMCID: PMC9352726 DOI: 10.1038/s41467-022-32218-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Accepted: 07/18/2022] [Indexed: 01/22/2023] Open
Abstract
Plague has caused three worldwide pandemics in history, including the Black Death in medieval ages. Yersinia pestis, the etiological agent of plague, has evolved a powerful arsenal to disrupt host immune defenses during evolution from enteropathogenic Y. pseudotuberculosis. Here, we find that two functionally redundant E3 ligase of Y. pestis, YspE1 and YspE2, can be delivered via type III secretion injectisome into host cytosol where they ubiquitinate multiple guanylate-binding proteins (GBPs) for proteasomal degradation. However, Y. pseudotuberculosis has no such capability due to lacking functional YspE1/2 homologs. YspE1/2-mediated GBP degradations significantly promote the survival of Y. pestis in macrophages and strongly inhibit inflammasome activation. By contrast, Gbpchr3−/−, chr5−/− macrophages exhibit much lowered inflammasome activation independent of YspE1/2, accompanied with an enhanced replication of Y. pestis. Accordingly, Gbpchr3−/−, chr5−/− mice are more susceptible to Y. pestis. We demonstrate that Y. pestis utilizes E3 ligases to subvert GBP-mediated host defense, which appears to be newly acquired by Y. pestis during evolution. Guanylate-binding proteins (GBPs) recognize pathogen containing vacuoles, leading to lysis of this intracellular niche and induction of inflammasomes. Here, Cao et al. show that Y. pestis, the causative agent of plague, secret two functionally redundant E3 ligase, YspE1 and YspE2, into the host’s cytosol to ubiquitinate multiple GBPs for proteasomal degradation to subvert host immune defense. This capability appears to be newly acquired by Y. pestis during evolution, since its closely related progenitor Y. pseudotuberculosis is unable to do so.
Collapse
Affiliation(s)
- Shiyang Cao
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yang Jiao
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Wei Jiang
- National Institute of Biological Sciences, Beijing, 102206, China
| | - Yarong Wu
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Si Qin
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yifan Ren
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yang You
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yafang Tan
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Xiao Guo
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Hongyan Chen
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yuan Zhang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Gengshan Wu
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Tong Wang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yazhou Zhou
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yajun Song
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Yujun Cui
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China
| | - Feng Shao
- National Institute of Biological Sciences, Beijing, 102206, China
| | - Ruifu Yang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China.
| | - Zongmin Du
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 100071, Beijing, China.
| |
Collapse
|
|
4
|
Ellner SP, Buchon N, Dörr T, Lazzaro BP. Host-pathogen immune feedbacks can explain widely divergent outcomes from similar infections. Proc Biol Sci 2021; 288:20210786. [PMID: 34034518 DOI: 10.1098/rspb.2021.0786] [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] [Indexed: 12/20/2022] Open
Abstract
A long-standing question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here, we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.
Collapse
Affiliation(s)
- Stephen P Ellner
- Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA.,Cornell Institute for Host-Microbe Interactions and Disease, Cornell University, Ithaca, NY 14853, USA
| | - Nicolas Buchon
- Department of Entomology, Cornell University, Ithaca, NY 14853, USA.,Cornell Institute for Host-Microbe Interactions and Disease, Cornell University, Ithaca, NY 14853, USA
| | - Tobias Dörr
- Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.,Cornell Institute for Host-Microbe Interactions and Disease, Cornell University, Ithaca, NY 14853, USA.,Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA
| | - Brian P Lazzaro
- Department of Entomology, Cornell University, Ithaca, NY 14853, USA.,Cornell Institute for Host-Microbe Interactions and Disease, Cornell University, Ithaca, NY 14853, USA
| |
Collapse
|
|
5
|
Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis. Proc Natl Acad Sci U S A 2020; 117:22984-22991. [PMID: 32868431 DOI: 10.1073/pnas.1917504117] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.
Collapse
|
|
6
|
Du J, Lin Z, Volovych O, Lu Z, Zou Z. A RhoGAP venom protein from Microplitis mediator suppresses the cellular response of its host Helicoverpa armigera. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2020; 108:103675. [PMID: 32173445 DOI: 10.1016/j.dci.2020.103675] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/10/2019] [Revised: 03/09/2020] [Accepted: 03/09/2020] [Indexed: 06/10/2023]
Abstract
Female parasitoid wasps normally inject virulence factors together with eggs into their host to counter host immunity defenses. A newly identified RhoGAP protein in the venom of Microplitis mediator compromises the cellular immunity of its host, Helicoverpa armigera. RhoGAP1 proteins entered H. armigera hemocytes, and the host cellular cytoskeleton was disrupted. Depletion of MmGAP1 by injection of dsRNA or antibody increased the wasp egg encapsulation rate. An immunoprecipitation assay of overexpressed MmGAP1 protein in a Helicoverpa cell line showed that MmGAP1 interacts with many cellular cytoskeleton associated proteins as well as Rho GTPases. A yeast two-hybrid and a pull-down assay demonstrated that MmGAP1 interacts with H. armigera RhoA and Cdc42. These results show that the RhoGAP protein in M. mediator can destroy the H. armigera hemocyte cellular cytoskeleton, restrain host cellular immune defense, and increase the probability of successful parasitism.
Collapse
Affiliation(s)
- Jie Du
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Zhe Lin
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Olga Volovych
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Zhiqiang Lu
- Department of Entomology, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, 712100, China
| | - Zhen Zou
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, 100049, China; Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, Huzhou University, Huzhou, 311300, China.
| |
Collapse
|
|
7
|
Difference in Strain Pathogenicity of Septicemic Yersinia pestis Infection in a TLR2 -/- Mouse Model. Infect Immun 2020; 88:IAI.00792-19. [PMID: 31907194 DOI: 10.1128/iai.00792-19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2019] [Accepted: 12/16/2019] [Indexed: 11/20/2022] Open
Abstract
Yersinia pestis is the causative agent of bubonic, pneumonic, and septicemic plague. We demonstrate that Toll-like receptor 2-deficient (TLR2-/-) mice are resistant to septicemic infection by the KIM5 strain of Y. pestis but not to infection by the CO92 Δpgm strain. This resistance is dependent on TLR2, the route of infection, and the isoform of YopJ. Elevated bacterial burdens were found in the spleens of CO92 Δpgm-infected animals by 24 h postinfection and in the livers by 4 days. The YopJ isoform present contributed directly to cytotoxicity and inflammatory cytokine production of bone marrow-derived macrophages from TLR2-/- mice. Immune cell trafficking is altered in CO92 Δpgm infections, with an increased neutrophil infiltration to the spleen 5 days postinfection. Immune cell infiltration to the liver was greater and earlier in KIM5-infected TLR2-/- mice. The functionality of the immune cells was assessed by the ability to develop reactive oxygen and nitrogen species. Our data suggest an inhibition of granulocytes in forming these species in CO92 Δpgm-infected TLR2-/- mice. These findings suggest that resistance to KIM5 in TLR2-/- mice is dependent on early immune cell trafficking and functionality.
Collapse
|
|
8
|
Bozcal E. A general view on virulence determinants and infection strategies of Yersinia enterocolitica. MINERVA BIOTECNOL 2020. [DOI: 10.23736/s1120-4826.19.02582-5] [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]
|
|
9
|
Milne-Davies B, Helbig C, Wimmi S, Cheng DWC, Paczia N, Diepold A. Life After Secretion- Yersinia enterocolitica Rapidly Toggles Effector Secretion and Can Resume Cell Division in Response to Changing External Conditions. Front Microbiol 2019; 10:2128. [PMID: 31572334 PMCID: PMC6753693 DOI: 10.3389/fmicb.2019.02128] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2019] [Accepted: 08/29/2019] [Indexed: 12/12/2022] Open
Abstract
Many pathogenic bacteria use the type III secretion system (T3SS) injectisome to manipulate host cells by injecting virulence-promoting effector proteins into the host cytosol. The T3SS is activated upon host cell contact, and its activation is accompanied by an arrest of cell division; hence, many species maintain a T3SS-inactive sibling population to propagate efficiently within the host. The enteric pathogen Yersinia enterocolitica utilizes the T3SS to prevent phagocytosis and inhibit inflammatory responses. Unlike other species, almost all Y. enterocolitica are T3SS-positive at 37°C, which raises the question, how these bacteria are able to propagate within the host, that is, when and how they stop secretion and restart cell division after a burst of secretion. Using a fast and quantitative in vitro secretion assay, we have examined the initiation and termination of type III secretion. We found that effector secretion begins immediately once the activating signal is present, and instantly stops when this signal is removed. Following effector secretion, the bacteria resume division within minutes after being introduced to a non-secreting environment, and the same bacteria are able to re-initiate effector secretion at later time points. Our results indicate that Y. enterocolitica use their type III secretion system to promote their individual survival when necessary, and are able to quickly switch their behavior toward replication afterwards, possibly gaining an advantage during infection.
Collapse
Affiliation(s)
| | | | | | | | | | - Andreas Diepold
- Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
| |
Collapse
|
|
10
|
Wang J, Yang B, An Y, Marquez-Lago T, Leier A, Wilksch J, Hong Q, Zhang Y, Hayashida M, Akutsu T, Webb GI, Strugnell RA, Song J, Lithgow T. Systematic analysis and prediction of type IV secreted effector proteins by machine learning approaches. Brief Bioinform 2019; 20:931-951. [PMID: 29186295 PMCID: PMC6585386 DOI: 10.1093/bib/bbx164] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2017] [Revised: 11/08/2017] [Indexed: 12/13/2022] Open
Abstract
In the course of infecting their hosts, pathogenic bacteria secrete numerous effectors, namely, bacterial proteins that pervert host cell biology. Many Gram-negative bacteria, including context-dependent human pathogens, use a type IV secretion system (T4SS) to translocate effectors directly into the cytosol of host cells. Various type IV secreted effectors (T4SEs) have been experimentally validated to play crucial roles in virulence by manipulating host cell gene expression and other processes. Consequently, the identification of novel effector proteins is an important step in increasing our understanding of host-pathogen interactions and bacterial pathogenesis. Here, we train and compare six machine learning models, namely, Naïve Bayes (NB), K-nearest neighbor (KNN), logistic regression (LR), random forest (RF), support vector machines (SVMs) and multilayer perceptron (MLP), for the identification of T4SEs using 10 types of selected features and 5-fold cross-validation. Our study shows that: (1) including different but complementary features generally enhance the predictive performance of T4SEs; (2) ensemble models, obtained by integrating individual single-feature models, exhibit a significantly improved predictive performance and (3) the 'majority voting strategy' led to a more stable and accurate classification performance when applied to predicting an ensemble learning model with distinct single features. We further developed a new method to effectively predict T4SEs, Bastion4 (Bacterial secretion effector predictor for T4SS), and we show our ensemble classifier clearly outperforms two recent prediction tools. In summary, we developed a state-of-the-art T4SE predictor by conducting a comprehensive performance evaluation of different machine learning algorithms along with a detailed analysis of single- and multi-feature selections.
Collapse
Affiliation(s)
- Jiawei Wang
- Biomedicine Discovery Institute and the Department of Microbiology at Monash University, Australia
| | - Bingjiao Yang
- National Engineering Research Center for Equipment and Technology of Cold Strip Rolling, College of Mechanical Engineering from Yanshan University, China
| | - Yi An
- College of Information Engineering, Northwest A&F University, China
| | - Tatiana Marquez-Lago
- Department of Genetics, University of Alabama at Birmingham (UAB) School of Medicine, USA
| | - André Leier
- Department of Genetics and the Informatics Institute, University of Alabama at Birmingham (UAB) School of Medicine, USA
| | - Jonathan Wilksch
- Department of Microbiology and Immunology at the University of Melbourne, Australia
| | | | - Yang Zhang
- Computer Science and Engineering in 2015 fromNorthwestern Polytechnical University, China
| | | | - Tatsuya Akutsu
- Bioinformatics Center, Institute for Chemical Research, Kyoto University, Japan
| | - Geoffrey I Webb
- Faculty of Information Technology, Monash Centre for Data Science, Monash University
| | - Richard A Strugnell
- Department of Microbiology and Immunology, Faculty of Medicine Dentistry and Health Sciences, University of Melbourne
| | - Jiangning Song
- Department of Biochemistry and Molecular Biology, Monash University, Australia
| | - Trevor Lithgow
- Department of Microbiology at Monash University, Australia
| |
Collapse
|
|
11
|
Mechanisms of Horizontal Cell-to-Cell Transfer of Wolbachia spp. in Drosophila melanogaster. Appl Environ Microbiol 2017; 83:AEM.03425-16. [PMID: 28087534 DOI: 10.1128/aem.03425-16] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Accepted: 01/10/2017] [Indexed: 12/18/2022] Open
Abstract
Wolbachia is an intracellular endosymbiont present in most arthropod and filarial nematode species. Transmission between hosts is primarily vertical, taking place exclusively through the female germ line, although horizontal transmission has also been documented. The results of several studies indicate that Wolbachia spp. can undergo transfer between somatic and germ line cells during nematode development and in adult flies. However, the mechanisms underlying horizontal cell-to-cell transfer remain largely unexplored. Here, we establish a tractable system for probing horizontal transfer of Wolbachia cells between Drosophila melanogaster cells in culture using fluorescence in situ hybridization (FISH). First, we show that horizontal transfer is independent of cell-to-cell contact and can efficiently take place through the culture medium within hours. Further, we demonstrate that efficient transfer utilizes host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Lastly, we provide evidence that this process is conserved between species, showing that horizontal transfer from mosquito to Drosophila cells takes place in a similar fashion. Altogether, our results indicate that Wolbachia utilizes host internalization machinery during infection, and this mechanism is conserved across insect species.IMPORTANCE Our work has broad implications for the control and treatment of tropical diseases. Wolbachia can confer resistance against a variety of human pathogens in mosquito vectors. Elucidating the mechanisms of horizontal transfer will be useful for efforts to more efficiently infect nonnatural insect hosts with Wolbachia as a biological control agent. Further, as Wolbachia is essential for the survival of filarial nematodes, understanding horizontal transfer might provide new approaches to treating human infections by targeting Wolbachia Finally, this work provides a key first step toward the genetic manipulation of Wolbachia.
Collapse
|
|
12
|
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.
Collapse
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
| |
Collapse
|
|
13
|
Stewart MK, Cookson BT. Evasion and interference: intracellular pathogens modulate caspase-dependent inflammatory responses. Nat Rev Microbiol 2016; 14:346-59. [PMID: 27174147 DOI: 10.1038/nrmicro.2016.50] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Pathogens have evolved to complete the virulence cycle of colonization, replication and dissemination in intimate association with a complex network of extracellular and intracellular surveillance systems that guard tissue spaces. In this Review, we discuss the strategies used by bacteria and viruses to evade or inhibit intracellular detection that is coupled to pro-inflammatory caspase-dependent protective responses. Such strategies include alterations of lipopolysaccharide (LPS) structures, the regulated expression of components of type III secretion systems, and the utilization of proteins that inhibit inflammasome formation, the enzymatic activity of caspases and cytokine signalling. Inflammation is crucial in response to exposure to pathogens, but is potentially damaging and thus tightly regulated. The threshold for the activation of pro-inflammatory caspases is determined by the immediate stimulus in the context of previous signals. Pathogen, genetic and situational factors modulate this threshold, which determines the ability of the host to resist infection while minimizing harm.
Collapse
Affiliation(s)
- Mary K Stewart
- Department of Microbiology, University of Washington, Seattle, Washington 98195, USA
| | - Brad T Cookson
- Department of Microbiology, University of Washington, Seattle, Washington 98195, USA.,Department of Laboratory Medicine, University of Washington, Seattle, Washington 98195, USA
| |
Collapse
|
|
14
|
Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence. Infect Immun 2016; 84:1062-1072. [PMID: 26810037 DOI: 10.1128/iai.00843-15] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2015] [Accepted: 01/19/2016] [Indexed: 02/06/2023] Open
Abstract
Pathogenic Yersinia species utilize a type III secretion system to translocate Yop effectors into infected host cells. Yop effectors inhibit innate immune responses in infected macrophages to promote Yersinia pathogenesis. In turn,Yersinia-infected macrophages respond to translocation of Yops by activating caspase-1, but different mechanisms of caspase-1 activation occur, depending on the bacterial genotype and the state of phagocyte activation. In macrophages activated with lipopolysaccharide (LPS) prior to Yersinia pseudotuberculosis infection, caspase-1 is activated by a rapid inflammasome-dependent mechanism that is inhibited by translocated YopM. The possibility that other effectors cooperate with YopM to inhibit caspase-1 activation in LPS-activated macrophages has not been investigated. Toward this aim, epistasis analysis was carried out in which the phenotype of aY. pseudotuberculosis yopM mutant was compared to that of a yopJ yopM, yopE yopM, yopH yopM, yopT yopM, or ypkA yopM mutant. Activation of caspase-1 was measured by cleavage of the enzyme, release of interleukin-1β (IL-1β), and pyroptosis in LPS-activated macrophages infected with wild-type or mutant Y. pseudotuberculosis strains. Results show enhanced activation of caspase-1 after infection with the yopJ yopM mutant relative to infection by any other single or double mutant. Similar results were obtained with the yopJ, yopM, and yopJ yopM mutants ofY ersinia pestis Following intravenous infection of mice, theY. pseudotuberculosis yopJ mutant was as virulent as the wild type, while the yopJ yopM mutant was significantly more attenuated than the yopM mutant. In summary, through epistasis analysis this work uncovered an important role for YopJ in inhibiting caspase-1 in activated macrophages and in promoting Yersinia virulence.
Collapse
|
|
15
|
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.
Collapse
|
|
16
|
Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis. Infect Immun 2015; 84:365-74. [PMID: 26553463 DOI: 10.1128/iai.01168-15] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Accepted: 11/02/2015] [Indexed: 02/03/2023] Open
Abstract
Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague.
Collapse
|
|
17
|
Chen Y, Duan R, Li X, Li K, Liang J, Liu C, Qiu H, Xiao Y, Jing H, Wang X. Homology analysis and cross-immunogenicity of OmpA from pathogenic Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. Mol Immunol 2015; 68:290-9. [PMID: 26435220 DOI: 10.1016/j.molimm.2015.09.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2015] [Revised: 09/15/2015] [Accepted: 09/22/2015] [Indexed: 11/19/2022]
Abstract
The outer membrane protein A (OmpA) is one of the intra-species conserved proteins with immunogenicity widely found in the family of Enterobacteriaceae. Here we first confirmed OmpA is conserved in the three pathogenic Yersinia: Yersinia pestis, Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica, with high homology at the nucleotide level and at the amino acid sequence level. The identity of ompA sequences for 262 Y. pestis strains, 134 Y. pseudotuberculosis strains and 219 pathogenic Y. enterocolitica strains are 100%, 98.8% and 97.7% similar. The main pattern of OmpA of pathogenic Yersinia are 86.2% and 88.8% identical at the nucleotide and amino acid sequence levels, respectively. Immunological analysis showed the immunogenicity of each OmpA and cross-immunogenicity of OmpA for pathogenic Yersinia where OmpA may be a vaccine candidate for Y. pestis and other pathogenic Yersinia.
Collapse
Affiliation(s)
- Yuhuang Chen
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Ran Duan
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Xu Li
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Kewei Li
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Junrong Liang
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Chang Liu
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Haiyan Qiu
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Yuchun Xiao
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Huaiqi Jing
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China
| | - Xin Wang
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing, China.
| |
Collapse
|
|
18
|
Vagima Y, Zauberman A, Levy Y, Gur D, Tidhar A, Aftalion M, Shafferman A, Mamroud E. Circumventing Y. pestis Virulence by Early Recruitment of Neutrophils to the Lungs during Pneumonic Plague. PLoS Pathog 2015; 11:e1004893. [PMID: 25974210 PMCID: PMC4431741 DOI: 10.1371/journal.ppat.1004893] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2014] [Accepted: 04/18/2015] [Indexed: 12/20/2022] Open
Abstract
Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated with a delayed immune response in the lungs. Because neutrophils are the first immune cells recruited to sites of infection, we investigated the mechanisms responsible for their delayed homing to the lung. During the first 24 hr after pulmonary infection with a fully virulent Y. pestis strain, no significant changes were observed in the lungs in the levels of neutrophils infiltrate, expression of adhesion molecules, or the expression of the major neutrophil chemoattractants keratinocyte cell-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor (G-CSF). In contrast, early induction of chemokines, rapid neutrophil infiltration and a reduced bacterial burden were observed in the lungs of mice infected with an avirulent Y. pestis strain. In vitro infection of lung-derived cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition of chemoattractants expression. However, the recruitment of neutrophils to the lungs of mice infected with the mutant was still delayed and associated with rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and G-CSF mRNA levels in the lungs were up-regulated early after infection with the mutant, their protein levels remained constant, suggesting that Y. pestis may employ additional mechanisms to suppress early chemoattractants induction in the lung. It therefore seems that prevention of the early influx of neutrophils to the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led to rapid homing of neutrophils to the lung followed by a reduction in bacterial counts at 24 hr post-infection and improved survival rates. These observations shed new light on the virulence mechanisms of Y. pestis during pneumonic plague, and have implications for the development of novel therapies against this pathogen.
Collapse
Affiliation(s)
- Yaron Vagima
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - Ayelet Zauberman
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - Yinon Levy
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - David Gur
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - Avital Tidhar
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - Moshe Aftalion
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - Avigdor Shafferman
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
| | - Emanuelle Mamroud
- Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel
- * E-mail:
| |
Collapse
|
|
19
|
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.
Collapse
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.
| |
Collapse
|
|
20
|
Choi EK, Kim SY, Kim SH, Paek YW, Kang IC. Proteolytic activity of Porphyromonas gingivalis attenuates MCP-1 mRNA expression in LPS-stimulated THP-1 cells. Microb Pathog 2014; 73:13-8. [DOI: 10.1016/j.micpath.2014.05.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2014] [Revised: 05/14/2014] [Accepted: 05/16/2014] [Indexed: 12/27/2022]
|
|
21
|
Li L, Yan H, Feng L, Li Y, Lu P, Hu Y, Chen S. LcrQ blocks the role of LcrF in regulating the Ysc-Yop type III secretion genes in Yersinia pseudotuberculosis. PLoS One 2014; 9:e92243. [PMID: 24658611 PMCID: PMC3962397 DOI: 10.1371/journal.pone.0092243] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2013] [Accepted: 02/18/2014] [Indexed: 11/18/2022] Open
Abstract
Pathogenic Yersinia species employ the Ysc-Yop type III secretion system (T3SS) encoded by a highly conserved pYV virulence plasmid to export the virulence effectors into host cells. The Ysc-Yop T3SS is tightly regulated by multiple contributing proteins that function at different levels. However, systematic transcriptional regulation analysis of Ysc-Yop T3SS is lacking and the detailed mechanism under this regulation process is still elusive. Aimed at systematically characterizing transcriptional regulations of all T3SS genes in Y. pseudotuberculosis, we amplified 97 non-coding fragments from the pYV plasmid and analyzed transcriptional responses of the T3SS genes under different growth conditions. Transcriptions of T3SS genes were induced at 37°C and genes encoding T3SS effectors were highly induced by further depletion of Ca2+. The temperature induced gene transcription process is mediated by modules encoded on the chromosome, while the Ca2+ depletion-induced process is controlled by the positive regulatory protein LcrF as well as the negative regulatory protein LcrQ. In this process, LcrQ shares the same targets with LcrF and the effect of LcrQ is dependent on the presence of LcrF. Furthermore, over-expression of LcrF showed the same phenotype as that of the lcrQ mutant strain and intracellular amount balance of LcrQ and LcrF is important in T3SS regulation. When the expression level of LcrF exceeds LcrQ, expression of the Ysc-Yop T3SS genes is activated and vice versa. Together, these data support a model in which LcrQ blocks the activation role of LcrF in regulating the transcription of T3SS genes in Yersinia.
Collapse
Affiliation(s)
- Lamei Li
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Huan Yan
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Lipeng Feng
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Yunlong Li
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Pei Lu
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
| | - Yangbo Hu
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- * E-mail: (YH); (SC)
| | - Shiyun Chen
- Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
- * E-mail: (YH); (SC)
| |
Collapse
|
|
22
|
Identification of secreted bacterial proteins by noncanonical amino acid tagging. Proc Natl Acad Sci U S A 2013; 111:433-8. [PMID: 24347637 DOI: 10.1073/pnas.1301740111] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
Collapse
|
|
23
|
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.
Collapse
|
|
24
|
Dallaire-Dufresne S, Barbeau X, Sarty D, Tanaka KH, Denoncourt AM, Lagüe P, Reith ME, Charette SJ. Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system. Microbiology (Reading) 2013; 159:1937-1945. [DOI: 10.1099/mic.0.067959-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Affiliation(s)
- Stéphanie Dallaire-Dufresne
- Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Quebec City, Quebec G1V 4G5, Canada
- Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
- Institut de biologie intégrative et des systèmes, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
| | - Xavier Barbeau
- Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
- Institut de biologie intégrative et des systèmes, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
| | - Darren Sarty
- Aquatic and Crop Resource Development, National Research Council Canada, Halifax, Nova Scotia B3H 3Z1, Canada
| | - Katherine H. Tanaka
- Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Quebec City, Quebec G1V 4G5, Canada
- Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
- Institut de biologie intégrative et des systèmes, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
| | - Alix M. Denoncourt
- Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Quebec City, Quebec G1V 4G5, Canada
- Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
- Institut de biologie intégrative et des systèmes, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
| | - Patrick Lagüe
- Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
- Institut de biologie intégrative et des systèmes, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
| | - Michael E. Reith
- Aquatic and Crop Resource Development, National Research Council Canada, Halifax, Nova Scotia B3H 3Z1, Canada
| | - Steve J. Charette
- Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Quebec City, Quebec G1V 4G5, Canada
- Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
- Institut de biologie intégrative et des systèmes, Faculté des sciences et de génie, Université Laval, Quebec City, Quebec G1V 0A6, Canada
| |
Collapse
|
|
25
|
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.
Collapse
Affiliation(s)
- Manuel Wolters
- Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
| | | | | | | | | | | | | | | |
Collapse
|
|
26
|
Immune evasion, immunopathology and the regulation of the immune system. Pathogens 2013; 2:71-91. [PMID: 25436882 PMCID: PMC4235712 DOI: 10.3390/pathogens2010071] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2012] [Revised: 02/06/2013] [Accepted: 02/07/2013] [Indexed: 12/26/2022] Open
Abstract
Costs and benefits of the immune response have attracted considerable attention in the last years among evolutionary biologists. Given the cost of parasitism, natural selection should favor individuals with the most effective immune defenses. Nevertheless, there exists huge variation in the expression of immune effectors among individuals. To explain this apparent paradox, it has been suggested that an over-reactive immune system might be too costly, both in terms of metabolic resources and risks of immune-mediated diseases, setting a limit to the investment into immune defenses. Here, we argue that this view neglects one important aspect of the interaction: the role played by evolving pathogens. We suggest that taking into account the co-evolutionary interactions between the host immune system and the parasitic strategies to overcome the immune response might provide a better picture of the selective pressures that shape the evolution of immune functioning. Integrating parasitic strategies of host exploitation can also contribute to understand the seemingly contradictory results that infection can enhance, but also protect from, autoimmune diseases. In the last decades, the incidence of autoimmune disorders has dramatically increased in wealthy countries of the northern hemisphere with a concomitant decrease of most parasitic infections. Experimental work on model organisms has shown that this pattern may be due to the protective role of certain parasites (i.e., helminths) that rely on the immunosuppression of hosts for their persistence. Interestingly, although parasite-induced immunosuppression can protect against autoimmunity, it can obviously favor the spread of other infections. Therefore, we need to think about the evolution of the immune system using a multidimensional trade-off involving immunoprotection, immunopathology and the parasitic strategies to escape the immune response.
Collapse
|
|
27
|
Tetratricopeptide repeat motifs in the world of bacterial pathogens: role in virulence mechanisms. Infect Immun 2012; 81:629-35. [PMID: 23264049 DOI: 10.1128/iai.01035-12] [Citation(s) in RCA: 118] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The tetratricopeptide repeat (TPR) structural motif is known to occur in a wide variety of proteins present in prokaryotic and eukaryotic organisms. The TPR motif represents an elegant module for the assembly of various multiprotein complexes, and thus, TPR-containing proteins often play roles in vital cell processes. As the TPR profile is well defined, the complete TPR protein repertoire of a bacterium with a known genomic sequence can be predicted. This provides a tremendous opportunity for investigators to identify new TPR-containing proteins and study them in detail. In the past decade, TPR-containing proteins of bacterial pathogens have been reported to be directly related to virulence-associated functions. In this minireview, we summarize the current knowledge of the TPR-containing proteins involved in virulence mechanisms of bacterial pathogens while highlighting the importance of TPR motifs for the proper functioning of class II chaperones of a type III secretion system in the pathogenesis of Yersinia, Pseudomonas, and Shigella.
Collapse
|
|
28
|
Kinetics of innate immune response to Yersinia pestis after intradermal infection in a mouse model. Infect Immun 2012; 80:4034-45. [PMID: 22966041 DOI: 10.1128/iai.00606-12] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
A hallmark of Yersinia pestis infection is a delayed inflammatory response early in infection. In this study, we use an intradermal model of infection to study early innate immune cell recruitment. Mice were injected intradermally in the ear with wild-type (WT) or attenuated Y. pestis lacking the pYV virulence plasmid (pYV(-)). The inflammatory responses in ear and draining lymph node samples were evaluated by flow cytometry and immunohistochemistry. As measured by flow cytometry, total neutrophil and macrophage recruitment to the ear in WT-infected mice did not differ from phosphate-buffered saline (PBS) controls or mice infected with pYV(-), except for a transient increase in macrophages at 6 h compared to the PBS control. Limited inflammation was apparent even in animals with high bacterial loads (10(5) to 10(6) CFU). In addition, activation of inflammatory cells was significantly reduced in WT-infected mice as measured by CD11b and major histocompatibility complex class II (MHC-II) expression. When mice infected with WT were injected 12 h later at the same intradermal site with purified LPS, Y. pestis did not prevent recruitment of neutrophils. However, significant reduction in neutrophil activation remained compared to that of PBS and pYV(-) controls. Immunohistochemistry revealed qualitative differences in neutrophil recruitment to the skin and draining lymph node, with WT-infected mice producing a diffuse inflammatory response. In contrast, focal sites of neutrophil recruitment were sustained through 48 h postinfection in pYV(-)-infected mice. Thus, an important feature of Y. pestis infection is reduced activation and organization of inflammatory cells that is at least partially dependent on the pYV virulence plasmid.
Collapse
|
|
29
|
ZHAO TONG, ZHAO PING, DOYLE MICHAELP. Detection and Isolation of Yersinia pestis Without Fraction 1 Antigen by Monoclonal Antibody in Foods and Water. J Food Prot 2012; 75:1555-61. [DOI: 10.4315/0362-028x.jfp-11-514] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Most available immunoassays for Yersinia pestis are based on the detection of fraction 1 antigen (F1) when yersiniae are grown at 37°C. A monoclonal antibody (MAb) was developed based on the detection of surface antigens that are not F1. F1-deficient Y. pestis cells were induced and used to immunize BALB/c mice from which MAb (immunoglobulin G1), which specifically recognizes Y. pestis, with or without F1, was obtained. This MAb (6B5) did not cross-react with enteric bacteria, including Yersinia enterocolitica. Enzyme-linked immunosorbent assay results revealed that MAb 6B5 is specific for Y. pestis, with the exception of a minor cross-reaction with Yersinia pseudotuberculosis. Western immunoblot analysis revealed that MAb 6B5 recognizes a Y. pestis outer membrane protein of ca. 30 kDa. Magnetic beads that were coated with MAb 6B5 were compared with beads coated with polyclonal antibody (PAb; rabbit) against Y. pestis for the isolation of Y. pestis in food and water samples by using a PATHATRIX cell concentration apparatus. Enrichment cultures of Y. pestis in different foods by using two different times (6 and 24 h) in brain heart infusion broth at 37°C were evaluated. Results revealed MAb 6B5–coated magnetic beads were equivalent to magnetic beads coated with PAb against Y. pestis A1122 whole cells in concentrating Y. pestis for isolation, especially when samples were enriched for 6 h. However, the selectivity for Y. pestis of the magnetic beads coated with MAb 6B5 was greater than that coated with PAb.
Collapse
Affiliation(s)
- TONG ZHAO
- Center for Food Safety, University of Georgia, Griffin, Georgia 30223-1797, USA
| | - PING ZHAO
- Center for Food Safety, University of Georgia, Griffin, Georgia 30223-1797, USA
| | - MICHAEL P. DOYLE
- Center for Food Safety, University of Georgia, Griffin, Georgia 30223-1797, USA
| |
Collapse
|
|
30
|
Rodríguez-Martín S, Kropp KA, Wilhelmi V, Lisnic VJ, Hsieh WY, Blanc M, Livingston A, Busche A, Tekotte H, Messerle M, Auer M, Fraser I, Jonjic S, Angulo A, Reddehase MJ, Ghazal P. Ablation of the regulatory IE1 protein of murine cytomegalovirus alters in vivo pro-inflammatory TNF-alpha production during acute infection. PLoS Pathog 2012; 8:e1002901. [PMID: 22952450 PMCID: PMC3431344 DOI: 10.1371/journal.ppat.1002901] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2012] [Accepted: 07/27/2012] [Indexed: 12/24/2022] Open
Abstract
Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo. The suppression of the production rather than the blockage of action of the potent inflammatory mediator TNFα is a particular hallmark of anti-TNFα mechanisms associated with microbial and parasitic infections. Whether this mode of counter-regulation is an important feature of infection by viruses is not clear. Also, it remains to be determined whether a specific pathogen gene in the context of an infection in vivo is capable of modulating levels of TNFα production. In this study we disclose a virus-mediated moderation of TNFα production, dependent on the ie1 gene of murine cytomegalovirus (MCMV). The ie1 gene product IE1 is a well-characterized nuclear protein capable of altering levels of host and viral gene expression although its biological role in the context of a natural infection is to date unknown. We provide evidence showing that ie1 is associated with a moderated pro-inflammatory cytokine response, in particular with TNFα production. Further, we show that the viral moderation of this cytokine is not only readily apparent in vitro but also in the natural host. The identification of a viral gene responsible for this mode of regulation in vivo may have therapeutic potential in the future in both anti-viral and anti-inflammatory strategies.
Collapse
Affiliation(s)
- Sara Rodríguez-Martín
- Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom
| | - Kai Alexander Kropp
- Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom
| | - Vanessa Wilhelmi
- Institute for Virology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Vanda Juranic Lisnic
- Department of Histology and Embryology/Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Wei Yuan Hsieh
- Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom
| | - Mathieu Blanc
- Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom
| | - Andrew Livingston
- Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom
| | - Andreas Busche
- Department of Virology, Hannover Medical School, Hannover, Germany
| | - Hille Tekotte
- Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
| | - Martin Messerle
- Department of Virology, Hannover Medical School, Hannover, Germany
| | - Manfred Auer
- University of Edinburgh, School of Biological Sciences (CSE) and School of Biomedical Sciences (CMVM), Edinburgh, United Kingdom
| | - Iain Fraser
- Laboratory of Systems Biology, National Institution of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Stipan Jonjic
- Department of Histology and Embryology/Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Ana Angulo
- Institut d'Investigacions Biomediques August Pi i Sunyer, Barcelona, Spain
| | - Matthias J. Reddehase
- Institute for Virology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Peter Ghazal
- Division of Pathway Medicine and Centre for Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom
- * E-mail:
| |
Collapse
|
|
31
|
Lountos GT, Tropea JE, Waugh DS. Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system. ACTA CRYSTALLOGRAPHICA. SECTION D, BIOLOGICAL CRYSTALLOGRAPHY 2012; 68:201-9. [PMID: 22349221 PMCID: PMC3282619 DOI: 10.1107/s0907444911054308] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/28/2011] [Accepted: 12/16/2011] [Indexed: 12/24/2022]
Abstract
The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.
Collapse
Affiliation(s)
- George T. Lountos
- Basic Science Program, SAIC-Frederick Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA
- Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA
| | - Joseph E. Tropea
- Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA
| | - David S. Waugh
- Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA
| |
Collapse
|
|
32
|
Haneda T, Ishii Y, Shimizu H, Ohshima K, Iida N, Danbara H, Okada N. Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection. Cell Microbiol 2012; 14:485-99. [PMID: 22188134 DOI: 10.1111/j.1462-5822.2011.01733.x] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through β-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.
Collapse
Affiliation(s)
- Takeshi Haneda
- Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo 108-8641, Japan
| | | | | | | | | | | | | |
Collapse
|
|
33
|
Yop effector proteins from Yersinia pseudotuberculosis impair dendritic cell activation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2012; 954:203-8. [PMID: 22782764 DOI: 10.1007/978-1-4614-3561-7_25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
|
|
34
|
Lu D, He P, Shan L. Bacterial effectors target BAK1-associated receptor complexes: One stone two birds. Commun Integr Biol 2011; 3:80-3. [PMID: 20585495 DOI: 10.4161/cib.3.2.10301] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2009] [Accepted: 10/11/2009] [Indexed: 11/19/2022] Open
Abstract
The long-standing association between hosts and microbes has generated some of most intricate relationships. The studies on molecular mechanisms of host-microbe interaction have been revealing many fascinating stories. Here we zoom in on a specific topic on the interplay between bacterial effectors and plant innate immune signaling. In particular, we will summarize our recent discovery that bacterial effector proteins, AvrPto and AvrPtoB, target plant immune signaling receptor complexes to interfere with host immune responses and development.
Collapse
|
|
35
|
Kaman WE, Hawkey S, van der Kleij D, Broekhuijsen MP, Silman NJ, Bikker FJ. A comprehensive study on the role of the Yersinia pestis virulence markers in an animal model of pneumonic plague. Folia Microbiol (Praha) 2011; 56:95-102. [PMID: 21468758 PMCID: PMC3109262 DOI: 10.1007/s12223-011-0027-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2010] [Accepted: 01/04/2011] [Indexed: 01/15/2023]
Abstract
We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF– strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.
Collapse
Affiliation(s)
- W E Kaman
- TNO Defence, Security and Safety, 2280 AA, Rijswijk, the Netherlands.
| | | | | | | | | | | |
Collapse
|
|
36
|
Biofilm development on Caenorhabditis elegans by Yersinia is facilitated by quorum sensing-dependent repression of type III secretion. PLoS Pathog 2011; 7:e1001250. [PMID: 21253572 PMCID: PMC3017118 DOI: 10.1371/journal.ppat.1001250] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2010] [Accepted: 12/06/2010] [Indexed: 12/30/2022] Open
Abstract
Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS. Many Gram-negative bacteria communicate by producing and sensing the presence of chemical signal molecules such as the N-acylhomoserine lactones (AHLs). Bacterial cells use AHLs to convey information about their environment, metabolism and population size. This type of chemical signalling is called ‘quorum sensing’ (QS) and is often used by pathogenic bacteria to promote acute or chronic infections through the control of motility, toxins, tissue degrading enzymes and surface-associated biofilms. Yersinia pseudotuberculosis is a human pathogen which forms biofilms on the surface of the nematode worm, Caenorhabditis elegans. This offers a simple means for investigating biofilm development on living tissues and can be used to identify genetic features of both the pathogen and the host that contribute to biofilm-associated infections. We have discovered that quorum sensing is required for Y. pseudotuberculosis biofilm formation on C. elegans through a regulatory pathway which involves the master motility regulator protein (FlhDC) reciprocally controlling bacterial swimming and the construction of a specialized secretion needle that delivers proteins into mammalian cells to disrupt their normal activities.
Collapse
|
|
37
|
van West P, de Bruijn I, Minor KL, Phillips AJ, Robertson EJ, Wawra S, Bain J, Anderson VL, Secombes CJ. The putative RxLR effector protein SpHtp1 from the fish pathogenic oomycete Saprolegnia parasitica is translocated into fish cells. FEMS Microbiol Lett 2010; 310:127-37. [PMID: 20659163 DOI: 10.1111/j.1574-6968.2010.02055.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
The fish pathogenic oomycete Saprolegnia parasitica causes the disease Saprolegniosis in salmonids and other freshwater fish, resulting in considerable economic losses in aquaculture. Very little is known about the molecular and cellular mechanisms underlying the infection process of fish pathogenic oomycetes. In order to investigate the interaction in detail, an in vitro infection assay using an Oncorhynchus mykiss (rainbow trout) cell line (RTG-2) was developed. In a zoospore/cyst cDNA library, we identified the ORF SpHtp1, which encodes a secreted protein containing an RxLR motif. Detailed expression analysis indicated that SpHtp1 is highly expressed in zoospores/cysts from S. parasitica and in the very early stages of infection on RTG-2 cells, when compared with in vitro-grown mycelium. Moreover, the protein, SpHtp1, was found to translocate into the RTG-2 trout cells, during the interaction with S. parasitica, and also when the RTG-2 cells were treated with recombinant SpHtp1 fused to a C-terminal His-tag. These findings suggest that protein translocation could play an important role in Saprolegniosis.
Collapse
Affiliation(s)
- Pieter van West
- Aberdeen Oomycete Laboratory, College of Life Sciences and Medicine, Institute of Medical Sciences, University of Aberdeen - School of Medical Sciences, Foresterhill, Aberdeen, UK.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
38
|
Three Yersinia pestis adhesins facilitate Yop delivery to eukaryotic cells and contribute to plague virulence. Infect Immun 2010; 78:4134-50. [PMID: 20679446 DOI: 10.1128/iai.00167-10] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.
Collapse
|
|
39
|
Wu B, Skarina T, Yee A, Jobin MC, DiLeo R, Semesi A, Fares C, Lemak A, Coombes BK, Arrowsmith CH, Singer AU, Savchenko A. NleG Type 3 effectors from enterohaemorrhagic Escherichia coli are U-Box E3 ubiquitin ligases. PLoS Pathog 2010; 6:e1000960. [PMID: 20585566 PMCID: PMC2891834 DOI: 10.1371/journal.ppat.1000960] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2010] [Accepted: 05/24/2010] [Indexed: 01/11/2023] Open
Abstract
NleG homologues constitute the largest family of Type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9' family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56+/-2 microM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.
Collapse
Affiliation(s)
- Bin Wu
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
| | - Tatiana Skarina
- Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada
| | - Adelinda Yee
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
| | - Marie-Claude Jobin
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
- Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada
| | - Rosa DiLeo
- Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada
| | - Anthony Semesi
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
| | - Christophe Fares
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
| | - Alexander Lemak
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
| | - Brian K. Coombes
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
| | - Cheryl H. Arrowsmith
- Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario, Canada
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
- Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada
| | - Alexander U. Singer
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
- Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada
| | - Alexei Savchenko
- Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
- Banting and Best Department for Medical Research, University of Toronto, C.H. Best Institute, Toronto, Ontario, Canada
- * E-mail:
| |
Collapse
|
|
40
|
Bi Y, Du Z, Yang H, Guo Z, Tan Y, Zhu Z, Yang R. Reduced apoptosis of mouse macrophages induced by yscW mutant of Yersinia pestis results from the reduced secretion of YopJ and relates to caspase-3 signal pathway. Scand J Immunol 2009; 70:358-67. [PMID: 19751270 DOI: 10.1111/j.1365-3083.2009.02297.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system (T3SS) that injects six Yersinia outer protein (Yop) effector proteins into the cytosol of macrophages, leading to disruption of host defence mechanisms. Here, we report that a T3SS structural protein YscW of Yersinia pestis contributed to the induction of apoptosis of murine macrophages. The apoptotic percentage of macrophages, from both mouse peritoneal cavity and spleen, and of RAW264.7 cell line, caused by the yscW mutant strain was significantly lower than that by wild type (WT) Y. pestis and yscW complemented strain. Meanwhile, detection of caspase-3 activity in macrophages, a key apoptosis-inducing protein, showed coincident results with the changes of macrophage apoptosis induced by WT, yscW mutant and complemented strains, indicating that macrophage apoptosis was related to caspase-3 signal pathways. However, ectopic expression of YscW in RAW264.7 cells cannot increase the macrophage apoptosis and death, suggesting that YscW itself could not induce macrophage apoptosis directly. To get insight into the mechanism of this phenomenon, we investigated the secretion of YopJ, which has been thought to be the only Yop effector related to apoptosis, in WT, mutant and complemented strains, respectively. Results showed that in yscW mutant strain, secretion of YopJ was decreased significantly in the supernatant than that in WT or complemented strain. This means although YscW does not induce apoptosis directly, it can indirectly affect apoptosis through reducing the secretion of YopJ.
Collapse
Affiliation(s)
- Y Bi
- Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing, China
| | | | | | | | | | | | | |
Collapse
|
|
41
|
Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2009; 16:1457-66. [PMID: 19710295 DOI: 10.1128/cvi.00172-09] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Yersinia pestis, the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naïve or gamma interferon (IFN-gamma)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis-infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-gamma and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-gamma impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-gamma. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-gamma-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naïve or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-gamma to survive and induce apoptosis in murine macrophages.
Collapse
|
|
42
|
Lountos GT, Austin BP, Nallamsetty S, Waugh DS. Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion. Protein Sci 2009; 18:467-74. [PMID: 19165725 DOI: 10.1002/pro.56] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 A). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a beta-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU.
Collapse
Affiliation(s)
- George T Lountos
- Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA
| | | | | | | |
Collapse
|
|
43
|
Srinivasan A, Nanton M, Griffin A, McSorley SJ. Culling of activated CD4 T cells during typhoid is driven by Salmonella virulence genes. THE JOURNAL OF IMMUNOLOGY 2009; 182:7838-45. [PMID: 19494308 DOI: 10.4049/jimmunol.0900382] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Pathogen-specific CD4 T cells are activated within a few hours of oral Salmonella infection and are essential for protective immunity. However, CD4 T cells do not participate in bacterial clearance until several weeks after infection, suggesting that Salmonella can inhibit or evade CD4 T cells that are activated at early time points. Here, we describe the progressive culling of initially activated CD4 T cells in Salmonella-infected mice. Loss of activated CD4 T cells was independent of early instructional programming, T cell precursor frequency, and Ag availability. In contrast, apoptosis of Ag-specific CD4 T cells was actively induced by live bacteria in a process that required Salmonella pathogenicity island-2 and correlated with increased expression of PD-L1. These data demonstrate efficient culling of initially activated Ag-specific CD4 cells by a microbial pathogen and document a novel strategy for bacterial immune evasion.
Collapse
Affiliation(s)
- Aparna Srinivasan
- Department of Medicine, Division of Gastroenterology, Center for Infectious Diseases and Microbiology Translational Research, McGuire Translational Research Facility, University of Minnesota Medical School, Minneapolis, MN 55455, USA
| | | | | | | |
Collapse
|
|
44
|
Tansini A, de Medeiros BMM. Susceptibility to Yersinia pseudotuberculosis infection is linked to the pattern of macrophage activation. Scand J Immunol 2009; 69:310-8. [PMID: 19284495 DOI: 10.1111/j.1365-3083.2008.02212.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
T helper 1 cells play a crucial role in the clearance of Yersinia pseudotuberculosis infection. By producing cytokines and presenting antigens to T cells, activated macrophages can orientate the adaptive immune response. The pathway used by macrophages to metabolize arginine has been employed as an important parameter to discriminate their activation state. In this study, the pattern of macrophage activation in Y. pseudotuberculosis-infected BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice and their immunostimulatory capacity were analysed. In the early phase of infection, macrophages obtained from C57BL/6 mice produced higher levels of NO, lower arginase activity, and larger amounts of IL-12 and TNF-alpha than macrophages from BALB/c mice. On the other hand, macrophages derived from BALB/c mice produced higher levels of IL-10 and TGF-beta than C57BL/6 mice. The Y. pseudotuberculosis infection leads to a fall in the macrophage immunostimulatory capacity of both strains of mice, with T-cell proliferation significantly reduced 12 h after infection. Moreover, we observed in the supernatant of co-culture of macrophages from infected mice with T lymphocytes from heat-killed Yersinia-immunized mice lower IFN-gamma production by cells from BALB/c mice than by C57BL/6 mice, and IL-4 was produced only by BALB/c mice on the first- and third-day post-infection. These results suggest that the pattern of macrophage activation is associated with susceptibility and resistance to Y. pseudotuberculosis infection in BALB/c and C57BL/6 mice.
Collapse
Affiliation(s)
- A Tansini
- Department of Biological Sciences, School of Pharmaceutical Sciences, UNESP-São Paulo State University, Araraquara, SP, Brazil
| | | |
Collapse
|
|
45
|
Roppenser B, Röder A, Hentschke M, Ruckdeschel K, Aepfelbacher M. Yersinia enterocolitica differentially modulates RhoG activity in host cells. J Cell Sci 2009; 122:696-705. [PMID: 19208761 DOI: 10.1242/jcs.040345] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Pathogenic bacteria of the genus Yersinia (Y. pestis, Y. enterocolitica and Y. pseudotuberculosis) have evolved numerous virulence factors (termed a stratagem) to manipulate the activity of Rho GTPases. Here, we show that Y. enterocolitica modulates RhoG, an upstream regulator of other Rho GTPases. At the contact site of virulent Y. enterocolitica and host cells, we could visualise spatiotemporally organised activation and deactivation of RhoG. On the one hand, the beta1-integrin clustering protein Invasin on the bacterial surface was found to activate RhoG and this promoted cell invasion. On the other hand, active RhoG was downregulated by the type III secretion system effector YopE acting as a GTPase-activating protein (GAP). YopE localised to Golgi and endoplasmic reticulum, and this determined its specificity for RhoG and other selected Rho GTPases. RhoG and its downstream effector module Elmo/Dock180 controlled both Rac1 activation by Invasin and Rac1 deactivation by YopE. We propose that RhoG is a central target of the Yersinia stratagem and a major upstream regulator of Rac1 during different phases of the Yersinia infection cycle.
Collapse
Affiliation(s)
- Bernhard Roppenser
- Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Hamburg-Eppendorf, Martinistrabetae 52, 20246 Hamburg, Germany
| | | | | | | | | |
Collapse
|
|
46
|
Sorci G, Faivre B. Inflammation and oxidative stress in vertebrate host-parasite systems. Philos Trans R Soc Lond B Biol Sci 2009; 364:71-83. [PMID: 18930878 DOI: 10.1098/rstb.2008.0151] [Citation(s) in RCA: 227] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Innate, inflammation-based immunity is the first line of vertebrate defence against micro-organisms. Inflammation relies on a number of cellular and molecular effectors that can strike invading pathogens very shortly after the encounter between inflammatory cells and the intruder, but in a non-specific way. Owing to this non-specific response, inflammation can generate substantial costs for the host if the inflammatory response, and the associated oxygen-based damage, get out of control. This imposes strong selection pressure that acts to optimize two key features of the inflammatory response: the timing of activation and resolution (the process of downregulation of the response). In this paper, we review the benefits and costs of inflammation-driven immunity. Our aim is to emphasize the importance of resolution of inflammation as a way of maintaining homeostasis against oxidative stress and to prevent the 'horror autotoxicus' of chronic inflammation. Nevertheless, host immune regulation also opens the way to pathogens to subvert host defences. Therefore, quantifying inflammatory costs requires assessing (i) short-term negative effects, (ii) delayed inflammation-driven diseases, and (iii) parasitic strategies to subvert inflammation.
Collapse
Affiliation(s)
- Gabriele Sorci
- BioGéoSciences, CNRS UMR 5561, Université de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France.
| | | |
Collapse
|
|
47
|
Essential role of the SycP chaperone in type III secretion of the YspP effector. J Bacteriol 2008; 191:1703-15. [PMID: 19114483 PMCID: PMC2648209 DOI: 10.1128/jb.01021-08] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The Ysa type III secretion (T3S) system enhances gastrointestinal infection by Yersinia enterocolitica bv. 1B. One effector protein targeted into host cells is YspP, a protein tyrosine phosphatase. It was determined in this study that the secretion of YspP requires a chaperone, SycP. Genetic analysis showed that deletion of sycP completely abolished the secretion of YspP without affecting the secretion of other Ysps by the Ysa T3S system. Analysis of the secretion and translocation signals of YspP defined the first 73 amino acids to form the minimal region of YspP necessary to promote secretion and translocation by the Ysa T3S system. Function of the YspP secretion/translocation signals was dependent on SycP. Curiously, when YspP was constitutively expressed in Y. enterocolitica bv. 1B, it was recognized and secreted by the Ysc T3S system and the flagellar T3S system. In these cases, the first 21 amino acids were sufficient to promote secretion, and while SycP did enhance secretion, it was not essential. However, neither the Ysc T3S system nor the flagellar T3S system translocated YspP into mammalian cells. This supports a model where SycP confers secretion/translocation specificities for YspP by the Ysa T3S system. A series of biochemical approaches further established that SycP specifically interacts with YspP and protected YspP degradation in the cell prior to secretion. Collectively, the evidence suggests that YspP secretion by the Ysa T3S system is a posttranslational event.
Collapse
|
|
48
|
Bi Y, Du Z, Han Y, Guo Z, Tan Y, Zhu Z, Yang R. Yersinia pestis and host macrophages: immunodeficiency of mouse macrophages induced by YscW. Immunology 2008; 128:e406-17. [PMID: 19191914 DOI: 10.1111/j.1365-2567.2008.02990.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system (T3SS) that transfers six Yersinia outer protein (Yop) effector proteins into the cytoplasm of eukaryotic cells, leading to disruption of host defence mechanisms. It is shown in this study that Yersinia pestis YscW, a protein of the T3SS injectisome, contributes to the induction of a deficiency in phagocytosis in host macrophages and a reduction in their antigen-presenting capacity. A Y. pestis strain lacking yscW had no effect on uptake by host macrophages. In mice infected with wild-type Y. pestis, the yscW mutant or a complement strain, immunodeficiency was observed in host macrophages compared with those from uninfected mice. However, the phagocytosis and antigen presenting capacities of macrophages infected by yscW mutant strain both in vivo and in vitro were significantly higher than those by wild type strain. Consistent with this finding, when YscW was expressed in the RAW264.7 macrophage cell line, phagocytosis and antigen-presenting capacities were significantly lower than those of the control groups. These results indicate that Y. pestis YscW may directly induce immunodeficiency in murine macrophages by crippling their phagocytosis and antigen-presenting capacities. These data provide evidences to Y. pestis pathogenesis that some proteins in T3SS injectisome, such as YscW protein, might play independent roles in disrupting host defense apart from their known functions.
Collapse
Affiliation(s)
- Yujing Bi
- State Key laboratory of Pathogen and Biosecurity, National Center for Biomedical Analysis, Army Center for Microbial Detection and Research, Institute of Microbiology and Epidemiology, Beijing, China
| | | | | | | | | | | | | |
Collapse
|
|
49
|
Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis. Proc Natl Acad Sci U S A 2008; 105:12497-502. [PMID: 18713860 DOI: 10.1073/pnas.0802773105] [Citation(s) in RCA: 96] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type III effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type III secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell.
Collapse
|
|
50
|
Tollenaere C, Rahalison L, Ranjalahy M, Rahelinirina S, Duplantier JM, Brouat C. CCR5 polymorphism and plague resistance in natural populations of the black rat in Madagascar. INFECTION GENETICS AND EVOLUTION 2008; 8:891-7. [PMID: 18703167 DOI: 10.1016/j.meegid.2008.07.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/28/2008] [Revised: 07/11/2008] [Accepted: 07/12/2008] [Indexed: 12/11/2022]
Abstract
Madagascar remains one of the world's largest plague foci. The black rat, Rattus rattus, is the main reservoir of plague in rural areas. This species is highly susceptible to plague in plague-free areas (low-altitude regions), whereas rats from the plague focus areas (central highlands) have evolved a disease-resistance polymorphism. We used the candidate gene CCR5 to investigate the genetic basis of plague resistance in R. rattus. We found a unique non-synonymous substitution (H184R) in a functionally important region of the gene. We then compared (i) CCR5 genotypes of dying and surviving plague-challenged rats and (ii) CCR5 allelic frequencies in plague focus and plague-free populations. Our results suggested a higher prevalence of the substitution in resistant animals compared to susceptible individuals, and a tendency for higher frequencies in plague focus areas compared to plague-free areas. Therefore, the CCR5 polymorphism may be involved in Malagasy black rat plague resistance. CCR5 and other undetermined plague resistance markers may provide useful biological information about host evolution and disease dynamics.
Collapse
Affiliation(s)
- C Tollenaere
- IRD UMR CBGP (INRA/IRD/Cirad/Montpellier SupAgro), Campus International Baillarguet, CS 30016, 34988 Montferrier sur Lez Cedex, France.
| | | | | | | | | | | |
Collapse
|