Chromosomally-Encoded Yersinia pestis Type III Secretion Effector Proteins Promote Infection in Cells and in Mice

ABSTRACT Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

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Chronic Effects of a Salmonella Type III Secretion Effector Protein AvrA In Vivo

PLoS ONE ◽

10.1371/journal.pone.0010505 ◽

2010 ◽

Vol 5 (5) ◽

pp. e10505 ◽

Cited By ~ 43

Author(s):

Rong Lu ◽

Shaoping Wu ◽

Xingyin Liu ◽

Yinglin Xia ◽

Yong-guo Zhang ◽

...

Keyword(s):

Type Iii Secretion ◽

Effector Protein ◽

Type Iii ◽

Chronic Effects ◽

Type Iii Secretion Effector

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Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

mBio ◽

10.1128/mbio.01459-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 31

Author(s):

Julia V. Monjarás Feria ◽

Matthew D. Lefebre ◽

York-Dieter Stierhof ◽

Jorge E. Galán ◽

Samuel Wagner

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Effector Proteins ◽

Switching Function ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Type Iii ◽

Autocatalytic Cleavage ◽

Serovar Typhimurium

ABSTRACTType III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of theSalmonella entericaserovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage eventper sedoes not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function.IMPORTANCEBacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

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Pseudomonas syringae Strains Naturally Lacking the Classical P. syringae hrp/hrc Locus Are Common Leaf Colonizers Equipped with an Atypical Type III Secretion System

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-2-0198 ◽

2010 ◽

Vol 23 (2) ◽

pp. 198-210 ◽

Cited By ~ 69

Author(s):

Christopher R. Clarke ◽

Rongman Cai ◽

David J. Studholme ◽

David S. Guttman ◽

Boris A. Vinatzer

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Plant Cells ◽

Type Iii Secretion System ◽

Ice Nucleation ◽

Secretion System ◽

Effector Proteins ◽

Population Densities ◽

Type Iii ◽

Effector Genes

Pseudomonas syringae is best known as a plant pathogen that causes disease by translocating immune-suppressing effector proteins into plant cells through a type III secretion system (T3SS). However, P. syringae strains belonging to a newly described phylogenetic subgroup (group 2c) are missing the canonical P. syringae hrp/hrc cluster coding for a T3SS, flanking effector loci, and any close orthologue of known P. syringae effectors. Nonetheless, P. syringae group 2c strains are common leaf colonizers and grow on some tested plant species to population densities higher than those obtained by other P. syringae strains on nonhost species. Moreover, group 2c strains have genes necessary for the production of phytotoxins, have an ice nucleation gene, and, most interestingly, contain a novel hrp/hrc cluster, which is only distantly related to the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster appears to encode a functional T3SS although the genes hrpK and hrpS, present in the classical P. syringae hrp/hrc cluster, are missing. The genome sequence of a representative group 2c strain also revealed distant orthologues of the P. syringae effector genes avrE1 and hopM1 and the P. aeruginosa effector genes exoU and exoY. A putative life cycle for group 2c P. syringae is discussed.

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Tandem Translation Generates a Chaperone for the Salmonella Type III Secretion System Protein SsaQ

Journal of Biological Chemistry ◽

10.1074/jbc.m111.278663 ◽

2011 ◽

Vol 286 (41) ◽

pp. 36098-36107 ◽

Cited By ~ 28

Author(s):

Xiu-Jun Yu ◽

Mei Liu ◽

Steve Matthews ◽

David W. Holden

Keyword(s):

Type Iii Secretion ◽

Ex Vivo ◽

Eukaryotic Cell ◽

Effector Proteins ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Function Loss ◽

Bacterial Cytoplasm

Type III secretion systems (T3SSs) of bacterial pathogens involve the assembly of a surface-localized needle complex, through which translocon proteins are secreted to form a pore in the eukaryotic cell membrane. This enables the transfer of effector proteins from the bacterial cytoplasm to the host cell. A structure known as the C-ring is thought to have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of the T3SS. Here, we studied SsaQ, an FliN-like putative C-ring protein of the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS. ssaQ produces two proteins by tandem translation: a long form (SsaQL) composed of 322 amino acids and a shorter protein (SsaQS) comprising the C-terminal 106 residues of SsaQL. SsaQL is essential for SPI-2 T3SS function. Loss of SsaQS impairs the function of the T3SS both ex vivo and in vivo. SsaQS binds to its corresponding region within SsaQL and stabilizes the larger protein. Therefore, SsaQL function is optimized by a novel chaperone-like protein, produced by tandem translation from its own mRNA species.

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Direct delivery of bacterial type III secretion system effector proteins to the eukaryotic cell as a tool to study bacterial pathogenesis

New Biotechnology ◽

10.1016/j.nbt.2009.06.917 ◽

2009 ◽

Vol 25 ◽

pp. S372

Author(s):

A. Jalili ◽

J. Atherton ◽

R. Delahay

Keyword(s):

Type Iii Secretion ◽

Type Iii Secretion System ◽

Eukaryotic Cell ◽

Bacterial Pathogenesis ◽

Secretion System ◽

Effector Proteins ◽

Type Iii ◽

Direct Delivery ◽

Bacterial Type

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Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton

Journal of Bacteriology ◽

10.1128/jb.01004-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 563-570 ◽

Cited By ~ 71

Author(s):

Andreas K. J. Veenendaal ◽

Charlotta Sundin ◽

Ariel J. Blocker

Keyword(s):

Type Iii Secretion ◽

Bacterial Infections ◽

Shigella Flexneri ◽

Effector Proteins ◽

Host Cells ◽

Incomplete Penetrance ◽

Secretion Systems ◽

Bacterial Surface ◽

Type Iii ◽

Type Iii Secretion Systems

ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

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Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis

Infection and Immunity ◽

10.1128/iai.71.5.2404-2413.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2404-2413 ◽

Cited By ~ 166

Author(s):

Sachiko Miyata ◽

Monika Casey ◽

Dara W. Frank ◽

Frederick M. Ausubel ◽

Eliana Drenkard

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Galleria Mellonella ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Triple Mutant ◽

Type Iii ◽

Wax Moth

ABSTRACT Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ΔexoU ΔexoY, ΔexoT ΔexoY, and ΔexoT ΔexoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ΔexoT ΔexoU ΔexoY triple mutant and a ΔpscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

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