Identification ofChlamydia trachomatisCT621, a protein delivered through the type III secretion system to the host cell cytoplasm and nucleus

Chlamydia trachomatis and Waddlia chondrophila are strict intracellular bacteria belonging to the Chlamydiales order. C. trachomatis is the most frequent bacterial cause of genital and ocular infections whereas W. chondrophila is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by C. trachomatis and 19 secreted by W. chondrophila. Using Y. enterocolitica as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two W. chondrophila T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to C. trachomatis class II chaperone. Furthermore, we identified a novel C. trachomatis effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.

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Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

mBio ◽

10.1128/mbio.02250-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 28

Author(s):

Pauline Basso ◽

Michel Ragno ◽

Sylvie Elsen ◽

Emeline Reboud ◽

Guillaume Golovkine ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Host Cell ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Iv Pili ◽

Clinical Strain ◽

Type Iii ◽

Type Iv ◽

Cell Membrane Disruption

ABSTRACT Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa . In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. IMPORTANCE The course and outcome of acute, toxigenic infections by Pseudomonas aeruginosa clinical isolates rely on the deployment of one of two virulence strategies: delivery of effectors by the well-known type III secretion system or the cytolytic activity of the recently identified two-partner secreted toxin, exolysin. Here, we characterize several features of the mammalian cell intoxication process mediated by exolysin. We found that exolysin requires the outer membrane protein ExlB for export into extracellular medium. Using in vitro recombinant protein and ex vivo assays, we demonstrated a pore-forming activity of exolysin. A cellular cytotoxicity screen of a transposon mutant library, made in an exolysin-producing clinical strain, identified type IV pili as bacterial appendages required for exolysin toxic function. This work deciphers molecular mechanisms underlying the activity of novel virulence factors used by P. aeruginosa clinical strains lacking the type III secretion system, including a requirement for the toxin-producing bacteria to be attached to the targeted cell to induce cytolysis, and defines new targets for developing antivirulence strategies.

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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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A bacterial type III secretion system inhibits actin polymerization to prevent pore formation in host cell membranes

The EMBO Journal ◽

10.1093/emboj/20.19.5373 ◽

2001 ◽

Vol 20 (19) ◽

pp. 5373-5382 ◽

Cited By ~ 93

Author(s):

G. I. Viboud

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Pore Formation ◽

Actin Polymerization ◽

Cell Membranes ◽

Type Iii Secretion System ◽

Secretion System ◽

Type Iii ◽

Host Cell Membranes ◽

Bacterial Type

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Bacterial adhesion and host cell factors leading to effector protein injection by type III secretion system

International Journal of Medical Microbiology ◽

10.1016/j.ijmm.2019.05.008 ◽

2019 ◽

Vol 309 (5) ◽

pp. 344-350 ◽

Cited By ~ 8

Author(s):

Erwin Bohn ◽

Michael Sonnabend ◽

Kristina Klein ◽

Ingo B. Autenrieth

Keyword(s):

Host Cell ◽

Bacterial Adhesion ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Protein ◽

Type Iii ◽

Host Cell Factors

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The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

mBio ◽

10.1128/mbio.01733-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 23

Author(s):

Lihi Shaulov ◽

Jenia Gershberg ◽

Wanyin Deng ◽

B. Brett Finlay ◽

Neta Sal-Man

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Bacterial Pathogens ◽

Type Iii Secretion System ◽

Secretion System ◽

Bacterial Virulence ◽

Effector Proteins ◽

Host Cells ◽

Gram Negative ◽

Type Iii

ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy.

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Transcriptional Induction of the Pseudomonas aeruginosa Type III Secretion System by Low Ca2+ and Host Cell Contact Proceeds through Two Distinct Signaling Pathways

Infection and Immunity ◽

10.1128/iai.00090-06 ◽

2006 ◽

Vol 74 (6) ◽

pp. 3334-3341 ◽

Cited By ~ 50

Author(s):

Nandini Dasgupta ◽

Alix Ashare ◽

Gary W. Hunninghake ◽

Timothy L. Yahr

Keyword(s):

Pseudomonas Aeruginosa ◽

Host Cell ◽

Type Iii Secretion ◽

Mammalian Cells ◽

Type Iii Secretion System ◽

Growth Conditions ◽

Secretion System ◽

Host Cells ◽

Dependent Manner ◽

Type Iii

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to intoxicate eukaryotic host cells. Transcription of the T3SS is induced under calcium-limited growth conditions or following intimate contact of P. aeruginosa with host cells. In the present study, we demonstrate that expression of the T3SS is controlled by two distinct regulatory mechanisms and that these mechanisms are differentially activated in a host cell-dependent manner. The first mechanism is dependent upon ExsC, a regulatory protein that couples transcription of the T3SS to the activity of the type III secretion machinery. ExsC is essential for induction of the T3SS under low-calcium-growth conditions and for T3SS-dependent cytotoxicity towards social amoebae, insect cells, and erythrocytes. The second regulatory mechanism functions independently of ExsC and is sufficient to elicit T3SS-dependent cytotoxicity towards certain types of mammalian cells. Although this second pathway (ExsC independent) is sufficient, an exsC mutant demonstrates a lag in the induction of cytotoxicity towards Chinese hamster ovary cells and is attenuated for virulence in a mouse pneumonia model. We propose that the ExsC-dependent pathway is required for full cytotoxicity towards all host cell types tested whereas the ExsC-independent pathway may represent an adaptation that allows P. aeruginosa to increase expression of the T3SS in response to specific types of mammalian cells.

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IpaD Localizes to the Tip of the Type III Secretion System Needle of Shigella flexneri

Infection and Immunity ◽

10.1128/iai.00440-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4391-4400 ◽

Cited By ~ 123

Author(s):

Marianela Espina ◽

Andrew J. Olive ◽

Roma Kenjale ◽

David S. Moore ◽

S. Fernando Ausar ◽

...

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Actin Polymerization ◽

Shigella Flexneri ◽

Type Iii Secretion System ◽

Secretion System ◽

Erythrocyte Membranes ◽

Host Cell Membrane ◽

Type Iii ◽

Surface Localization

ABSTRACT Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes.

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The Role of the Membrane-Associated Domain of the Export Apparatus Protein, EscV (SctV), in the Activity of the Type III Secretion System

Frontiers in Microbiology ◽

10.3389/fmicb.2021.719469 ◽

2021 ◽

Vol 12 ◽

Author(s):

Boško Mitrović ◽

Shir Lezerovich ◽

Neta Sal-Man

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Health Concern ◽

Loss Of Function ◽

Reporter System ◽

Host Cell Membrane ◽

Type Iii

Diarrheal diseases remain a major public health concern worldwide. Many of the causative bacterial pathogens that cause these diseases have a specialized protein complex, the type III secretion system (T3SS), which delivers effector proteins directly into host cells. These effectors manipulate host cell processes for the benefit of the infecting bacteria. The T3SS structure resembles a syringe anchored within the bacterial membrane, projecting toward the host cell membrane. The entry port of the T3SS substrates, called the export apparatus, is formed by five integral membrane proteins. Among the export apparatus proteins, EscV is the largest, and as it forms a nonamer, it constitutes the largest portion of the export apparatus complex. While there are considerable data on the soluble cytoplasmic domain of EscV, our knowledge of its membrane-associated section and its transmembrane domains (TMDs) is still very limited. In this study, using an isolated genetic reporter system, we found that TMD5 and TMD6 of EscV mediate strong self-oligomerization. Substituting these TMDs within the full-length protein with a random hydrophobic sequence resulted in a complete loss of function of the T3SS, further suggesting that the EscV TMD5 and TMD6 sequences have a functional role in addition to their structural role as membrane anchors. As we observed only mild reduction in the ability of the TMD-exchanged variants to integrate into the full or intermediate T3SS complexes, we concluded that EscV TMD5 and TMD6 are not crucial for the global assembly or stability of the T3SS complex but are rather involved in promoting the necessary TMD–TMD interactions within the complex and the overall TMD orientation to allow channel opening for the entry of T3SS substrates.

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