Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types

Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

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Novel Type III Effectors in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00161-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 18

Author(s):

David Burstein ◽

Shirley Satanower ◽

Michal Simovitch ◽

Yana Belnik ◽

Meital Zehavi ◽

...

Keyword(s):

Machine Learning ◽

Pseudomonas Aeruginosa ◽

Host Cell ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Content Type ◽

Machine Learning Classification ◽

Type Iii ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes chronic and acute infections in immunocompromised patients. Most P. aeruginosa strains encode an active type III secretion system (T3SS), utilized by the bacteria to deliver effector proteins from the bacterial cell directly into the cytoplasm of the host cell. Four T3SS effectors have been discovered and extensively studied in P. aeruginosa: ExoT, ExoS, ExoU, and ExoY. This is especially intriguing in light of P. aeruginosa's ability to infect a wide range of hosts. We therefore hypothesized that additional T3SS effectors that have not yet been discovered are encoded in the genome of P. aeruginosa. Here, we applied a machine learning classification algorithm to identify novel P. aeruginosa effectors. In this approach, various types of data are integrated to differentiate effectors from the rest of the open reading frames of the bacterial genome. Due to the lack of a sufficient learning set of positive effectors, our machine learning algorithm integrated genomic information from another Pseudomonas species and utilized dozens of features accounting for various aspects of the effector coding genes and their products. Twelve top-ranking predictions were experimentally tested for T3SS-specific translocation, leading to the discovery of two novel T3SS effectors. We demonstrate that these effectors are not part of the injection structural complex and report initial efforts toward their characterization. IMPORTANCE Pseudomonas aeruginosa uses a type III secretion system (T3SS) to secrete toxic proteins, termed effectors, directly into the cytoplasm of the host cell. The activation of this secretion system is correlated with disease severity and patient death. Compared with many other T3SS-utilizing pathogenic bacteria, P. aeruginosa has a fairly limited arsenal of effectors that have been identified. This is in sharp contrast with the wide range of hosts that this bacterium can infect. The discovery of two novel effectors described here is an important step toward better understanding of the virulence and host evasion mechanisms adopted by this versatile pathogen and may provide novel approaches to treat P. aeruginosa infections.

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Autophagy is induced by the type III secretion system of Vibrio alginolyticus in several mammalian cell lines

Archives of Microbiology ◽

10.1007/s00203-010-0646-9 ◽

2010 ◽

Vol 193 (1) ◽

pp. 53-61 ◽

Cited By ~ 24

Author(s):

Zhe Zhao ◽

Lvping Zhang ◽

Chunhua Ren ◽

Jingjing Zhao ◽

Chang Chen ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cell ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Vibrio Alginolyticus ◽

Type Iii ◽

Mammalian Cell Lines

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Downregulation of Mitogen-Activated Protein Kinases by the Bordetella bronchiseptica Type III Secretion System Leads to Attenuated Nonclassical Macrophage Activation

Infection and Immunity ◽

10.1128/iai.73.1.308-316.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 308-316 ◽

Cited By ~ 10

Author(s):

Annette Reissinger ◽

Jason A. Skinner ◽

Ming H. Yuk

Keyword(s):

Dendritic Cells ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Cell Types ◽

Mapk Signaling ◽

Secretion System ◽

Bordetella Bronchiseptica ◽

Mitogen Activated Protein Kinase ◽

Type Iii ◽

Mitogen Activated Protein

ABSTRACT Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-κB in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.

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Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines

Microbiology ◽

10.1099/mic.0.024919-0 ◽

2009 ◽

Vol 155 (3) ◽

pp. 837-851 ◽

Cited By ~ 27

Author(s):

Xiaohui Zhou ◽

Michael E. Konkel ◽

Douglas R. Call

Keyword(s):

Cell Death ◽

Cell Lines ◽

Type Iii Secretion ◽

Vibrio Parahaemolyticus ◽

Active Form ◽

Type Iii Secretion System ◽

Secretion System ◽

Monocytic Cell ◽

Type Iii ◽

System 1

The Vibrio parahaemolyticus type III secretion system 1 (T3SS1) induces cytotoxicity in mammalian epithelial cells. We characterized the cell death phenotype in both epithelial (HeLa) and monocytic (U937) cell lines following infection with V. parahaemolyticus. Using a combination of the wild-type strain and gene knockouts, we confirmed that V. parahaemolyticus strain NY-4 was able to induce cell death in both cell lines via a T3SS1-dependent mechanism. Bacterial contact, but not internalization, was required for T3SS1-induced cytotoxicity. The mechanism of cell death involves formation of a pore structure on the surface of infected HeLa and U937 cells, as demonstrated by cellular swelling, uptake of cell membrane-impermeable dye and protection of cytotoxicity by osmoprotectant (PEG3350). Western blot analysis showed that poly ADP ribose polymerase (PARP) was not cleaved and remained in its full-length active form. This result was evident for seven different V. parahaemolyticus strains. V. parahaemolyticus-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) or the caspase-1 inhibitor N-acetyl-tyrosyl-valyl-alanyl-aspartyl-aldehyde (Ac-YVAD-CHO); thus, caspases were not involved in T3SS1-induced cytotoxicity. DNA fragmentation was not evident following infection and autophagic vacuoles were not observed after monodansylcadaverine staining. We conclude that T3SS1 of V. parahaemolyticus strain NY-4 induces a host cell death primarily via oncosis rather than apoptosis, pyroptosis or autophagy.

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Identification of a Novel SalmonellaInvasion Locus Homologous to Shigella ipgDE

Journal of Bacteriology ◽

10.1128/jb.180.7.1793-1802.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1793-1802 ◽

Cited By ~ 98

Author(s):

K. Heran Hong ◽

Virginia L. Miller

Keyword(s):

Tissue Culture ◽

Type Iii Secretion ◽

Salmonella Enteritidis ◽

Chinese Hamster ◽

Type Iii Secretion System ◽

Pathogenicity Island ◽

Secretion System ◽

Virulence Plasmid ◽

Type Iii ◽

Culture Supernatants

ABSTRACT Genes essential for Salmonella typhimurium invasion have been localized to Salmonella pathogenicity island 1 (SPI1) on the chromosome. However, it is clear that other genes are required for the invasion process. Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells. Two invasion defective mutants were isolated by screening 2,500 Tn10dTc insertion mutants of S. typhimurium in the tissue culture invasion assay. One of the invasion mutants, SVM167, has an insertion between centisomes 24.5 and 25.5 in an operon homologous to theipgDEF operon of the Shigella flexneri andShigella sonnei virulence plasmid. A second mutant, SVM168, has an insertion in an IS3-type element with homology to the Salmonella enteritidis IS1351 element andYersinia enterocolitica IS1400 element from a high-pathogenicity island. Further characterization of SVM167 showed that culture supernatants from this mutant lack a previously uncharacterized protein that is also missing from culture supernatants of a SPI1 mutant, suggesting it can be secreted by the SPI1 type III secretion system. In addition, transcription of this operon,sigDE (Salmonella invasion gene), is dependent on the presence of sirA, an activator of hilAexpression. HilA activates transcription of several of the SPI1 genes but does not appear to have a major role in activation of transcription from the sigDE promoter.

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Killing from the inside: Intracellular role of T3SS in the fate ofPseudomonas aeruginosawithin macrophages revealed bymgtCandoprFmutants

10.1101/389718 ◽

2018 ◽

Author(s):

Preeti Garai ◽

Laurence Berry ◽

Malika Moussouni ◽

Sophie Bleves ◽

Anne-Béatrice Blanc-Potard

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Gene Expression Pattern ◽

Type Iii Secretion System ◽

Cell Types ◽

Cell Lysis ◽

Secretion System ◽

Intracellular Bacteria ◽

Type Iii

AbstractWhile considered solely an extracellular pathogen, increasing evidence indicates thatPseudomonas aeruginosaencounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-typeP. aeruginosaPAO1 strain by live and electron microscopy.P. aeruginosafirst resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis. Two bacterial factors, MgtC and OprF, recently identified to be important for survival ofP. aeruginosain macrophages, were found to be involved in bacterial escape from the phagosome as well as cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes ofP. aeruginosawere down-regulated within macrophages in bothmgtCandoprFmutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern ofmgtCandoprFmutants, a T3SS mutant(ΔpscN)exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellularP. aeruginosatowards macrophages. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life ofP. aeruginosa, allowing internalized bacteria to evade macrophages.Author summaryThe ability of professional phagocytes to ingest and kill microorganisms is central to host defense andPseudomonas aeruginosahas developed mechanisms to avoid being killed by phagocytes. While considered an extracellular pathogen,P. aeruginosahas been reported to be engulfed by macrophages in animal models. Here, we visualized the fate ofP. aeruginosawithin cultured macrophages, revealing macrophage lysis driven by intracellularP. aeruginosa. Two bacterial factors, MgtC and OprF, recently discovered to be involved in the intramacrophage survival ofP. aeruginosa, appeared to play role in this cytotoxicity caused by intracellular bacteria. We provided evidence that type III secretion system (T3SS) gene expression is lowered intracellularly inmgtCandoprFmutants. We further showed that intramacrophageP. aeruginosauses its T3SS, specifically the ExoS effector, to promote phagosomal escape and cell lysis. We thus describe a transient intramacrophage stage ofP. aeruginosathat could contribute to bacterial dissemination.

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Salmonella Pathogenicity Island 2-Encoded Proteins SseC and SseD Are Essential for Virulence and Are Substrates of the Type III Secretion System

Infection and Immunity ◽

10.1128/iai.69.2.737-743.2001 ◽

2001 ◽

Vol 69 (2) ◽

pp. 737-743 ◽

Cited By ~ 43

Author(s):

Joanna R. Klein ◽

Bradley D. Jones

Keyword(s):

Type Iii Secretion ◽

Type Iii Secretion System ◽

Systemic Infection ◽

Subcellular Location ◽

Pathogenicity Island ◽

Secretion System ◽

Bacterial Membrane ◽

Phagocytic Cells ◽

Type Iii ◽

Bacterial Cell Membrane

ABSTRACT Survival of Salmonella enterica serovar Typhimurium within host phagocytic cells is a critical step in establishing systemic infection in mice. Genes within Salmonellapathogenicity island 2 (SPI-2) encode a type III secretion system that is required for establishment of systemic infection. Several proteins encoded by SPI-2 have homology to type III secreted proteins from enteropathogenic Escherichia coli and Yersiniaand, based on that homology, are predicted to be secreted through the SPI-2 type III secretion system. We have investigated the roles of two of these proteins, SseC and SseD. We demonstrate here that the SseD protein is required for systemic Salmonella infection of the mouse, and we confirmed the virulence requirement for the SseC protein. Experiments were performed, using cellular fractionation and immunoblotting, to identify the subcellular location of the SseC and SseD proteins. Both proteins were found to localize predominantly to the bacterial cell membrane. In addition, our work revealed that SseC and SseD are exposed to the extracellular environment and are loosely associated with the bacterial membrane. Furthermore, localization of SseC and SseD to the bacterial membrane was found to require a functional SPI-2 type III secretion system. Collectively, these results indicate that the SseC and SseD proteins are secreted by the SPI-2 type III secretion system to the bacterial membrane in order to perform their virulence functions.

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