CesD2 of Enteropathogenic Escherichia coli Is a Second Chaperone for the Type III Secretion Translocator Protein EspD

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

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A Degenerate Type III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis

Journal of Bacteriology ◽

10.1128/jb.187.23.8164-8171.2005 ◽

2005 ◽

Vol 187 (23) ◽

pp. 8164-8171 ◽

Cited By ~ 44

Author(s):

Diana Ideses ◽

Uri Gophna ◽

Yossi Paitan ◽

Roy R. Chaudhuri ◽

Mark J. Pallen ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Gene Clusters ◽

Secretion System ◽

Effector Proteins ◽

E Coli ◽

Type Iii

ABSTRACT The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2sepsis, which, although degenerate, contributes to pathogenesis. ETT2sepsis has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.

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Mutations in Salmonella Pathogenicity Island 2 (SPI2) Genes Affecting Transcription of SPI1 Genes and Resistance to Antimicrobial Agents

Journal of Bacteriology ◽

10.1128/jb.180.18.4775-4780.1998 ◽

1998 ◽

Vol 180 (18) ◽

pp. 4775-4780 ◽

Cited By ~ 74

Author(s):

Jörg Deiwick ◽

Thomas Nikolaus ◽

Jaqueline E. Shea ◽

Colin Gleeson ◽

David W. Holden ◽

...

Keyword(s):

Type Iii Secretion ◽

Antimicrobial Agents ◽

Type Iii Secretion System ◽

Polymyxin B ◽

Secretion System ◽

Effector Proteins ◽

Secretion Systems ◽

Type Iii ◽

Genes Encoding

ABSTRACT The Salmonella typhimurium genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of S. typhimurium to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of sipC,prgK, and hilA, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.

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SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01859-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2863-2872 ◽

Cited By ~ 27

Author(s):

Jay L. Mellies ◽

Kenneth R. Haack ◽

Derek C. Galligan

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Host Cells ◽

Watery Diarrhea ◽

Type Iii ◽

Lexa Protein

ABSTRACT Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. Enteropathogenic Escherichia coli (EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that the LEE2 and LEE3 operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlapping LEE2/LEE3 promoters. Expression of the lexA1 allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-located nleA gene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells.

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Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

Microbiology ◽

10.1099/mic.0.044461-0 ◽

2011 ◽

Vol 157 (5) ◽

pp. 1428-1445 ◽

Cited By ~ 60

Author(s):

Devendra H. Shah ◽

Xiaohui Zhou ◽

Tarek Addwebi ◽

Margaret A. Davis ◽

Lisa Orfe ◽

...

Keyword(s):

Type Iii Secretion ◽

Salmonella Enterica ◽

Cell Model ◽

Surrogate Marker ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Type Iii ◽

Cell Invasiveness

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins.

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Differential expression of Salmonella type III secretion system factors InvJ, PrgJ, SipC, SipD, SopA and SopB in cultures and in mice

Microbiology ◽

10.1099/mic.0.032318-0 ◽

2010 ◽

Vol 156 (1) ◽

pp. 116-127 ◽

Cited By ~ 19

Author(s):

Hao Gong ◽

Gia-Phong Vu ◽

Yong Bai ◽

Edward Yang ◽

Fenyong Liu ◽

...

Keyword(s):

Differential Expression ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Systemic Infection ◽

Secretion System ◽

Flag Epitope ◽

Type Iii ◽

Late Stages

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.

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SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system

Microbiology ◽

10.1099/mic.0.26190-0 ◽

2003 ◽

Vol 149 (5) ◽

pp. 1103-1111 ◽

Cited By ~ 30

Author(s):

Javier Ruiz-Albert ◽

Rosanna Mundy ◽

Xiu-Jun Yu ◽

Carmen R. Beuzón ◽

David W. Holden

Keyword(s):

Type Iii Secretion ◽

Sequence Similarity ◽

Coiled Coil ◽

Type Iii Secretion System ◽

Pathogenicity Island ◽

Secretion System ◽

Effector Proteins ◽

Type Iii ◽

Virulence Attenuation

The type III secretion system (TTSS) encoded by the Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages and for systemic infection in mice. Many TTSS secreted proteins, including effectors and components of the translocon, require chaperones which promote their stability, prevent their premature interactions or facilitate their secretion. In this study, the function of the first gene (sseA) of one of the SPI-2 operons (sseA–G) was investigated. This operon includes genes that encode translocon components (SseB, SseC and SseD), translocated proteins (SseF and SseG) and putative chaperones (SscA and SscB). sseA encodes a 12·5 kDa protein with a C-terminal region with the potential to form a coiled-coil structure, but no sequence similarity to other proteins. Mutation of sseA results in severe virulence attenuation and an intracellular replication defect. It is shown here that SseA is not a secreted protein, but is required for SPI-2-dependent translocation of two effector proteins (SifA and PipB). Furthermore, the translocon components SseB and SseD were not detected in an sseA mutant strain. By using a yeast two-hybrid assay and column binding experiments, it is demonstrated that SseA interacts directly with SseB and SseD. These results indicate that SseA is a chaperone for SseB and SseD. The inability of an sseA mutant to assemble the SPI-2 TTSS translocon accounts for its high level of virulence attenuation in vivo. To the authors' knowledge, this is the first chaperone described for the SPI-2 TTSS.

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Chaperone mediated coupling of subunit availability to activation of flagellar Type III Secretion

10.1101/2020.01.10.902387 ◽

2020 ◽

Author(s):

Owain J. Bryant ◽

Betty Y-W. Chung ◽

Gillian M. Fraser

Keyword(s):

Cell Membrane ◽

Electric Potential ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Proton Motive Force ◽

Secretion System ◽

Motive Force ◽

Type Iii

AbstractBacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilise the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf-driven export by the fT3SS.

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In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

Infection and Immunity ◽

10.1128/iai.69.12.7254-7261.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7254-7261 ◽

Cited By ~ 35

Author(s):

Carmen R. Beuzón ◽

Kate E. Unsworth ◽

David W. Holden

Keyword(s):

Virulence Factors ◽

Type Iii Secretion ◽

Salmonella Enterica ◽

Type Iii Secretion System ◽

Pathogenicity Island ◽

Secretion System ◽

Type Iii ◽

Serovar Typhimurium

ABSTRACT Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection byS. enterica serovar Typhimurium: theSalmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently toS. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.

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Novel Effector Protein EspY3 of Type III Secretion System from Enterohemorrhagic Escherichia coli Is Localized in Actin Pedestals

Microorganisms ◽

10.3390/microorganisms6040112 ◽

2018 ◽

Vol 6 (4) ◽

pp. 112 ◽

Cited By ~ 2

Author(s):

Mariano Larzábal ◽

Wanderson Marques Da Silva ◽

Nahuel Riviere ◽

Ángel Cataldi

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Effector Proteins ◽

Enterohemorrhagic Escherichia Coli ◽

Effector Protein ◽

Type Iii ◽

T3ss Effector ◽

Intestinal Enterocytes

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC) are attaching and effacing (A/E) pathogens, which translocate effector proteins to intestinal enterocytes through a type III secretion system (T3SS). T3SS and most of its effector proteins are encoded in a pathogenicity island called LEE. Recently, new effectors have been located outside the LEE. This study aimed to characterize EspY3, a novel non-LEE encoded T3SS effector of EHEC. EspY3 shares homology with SopD and PipB2 effector proteins of Salmonella’s T3SS-1 and T3SS-2, respectively. The presence of recombinant EspY3 in the supernatant samples demonstrated that EspY3 was secreted by the T3SS of EHEC and EPEC. Through infection assays, we demonstrated the translocation of EspY3 into Caco-2 cells by T3SS of EPEC. The subcellular localization of EspY3 was determined in the pedestal region, where its presence generates a significant increase in the size of the pedestals area. The EspY3 effector induced the elongation of polymerized actin pedestals in infected Caco-2 by EPEC. This study confirmed that EspY3 is part of the repertoire of T3SS effectors of EHEC O157:H7, and that it participates in modeling cellular actin during the infection.

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Expression of and secretion through the Aeromonas salmonicida type III secretion system

Microbiology ◽

10.1099/mic.0.28485-0 ◽

2006 ◽

Vol 152 (5) ◽

pp. 1275-1286 ◽

Cited By ~ 29

Author(s):

Roger O. Ebanks ◽

Leah C. Knickle ◽

Michel Goguen ◽

Jessica M. Boyd ◽

Devanand M. Pinto ◽

...

Keyword(s):

Growth Temperature ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Growth Conditions ◽

Secretion System ◽

Aeromonas Salmonicida ◽

Effector Proteins ◽

Type Iii ◽

Low Calcium

Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 °C, but not at its more natural growth temperature of 17 °C. More modest increases in expression occur at 24 °C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 °C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 °C in salt concentrations ranging from 0·19 to 0·38 M NaCl. It is also shown that growth at 28 °C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.

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