Enteropathogenic Escherichia coli Type III Effectors EspG and EspG2 Alter Epithelial Paracellular Permeability

ABSTRACT Enteropathogenic Escherichia coli (EPEC) delivers a subset of effectors into host cells via a type III secretion system. Here we show that the type III effector EspG and its homologue EspG2 alter epithelial paracellular permeability. When MDCK cells were infected with wild-type (WT) EPEC, RhoA was activated, and this event was dependent on the delivery of either EspG or EspG2 into host cells. In contrast, a loss of transepithelial electrical resistance and ZO-1 disruption were induced by infection with an espG/espG2 double-knockout mutant, as was the case with the WT EPEC, indicating that EspG/EspG2 is not involved in the disruption of tight junctions during EPEC infection. Although EspG- and EspG2-expressing MDCK cells exhibited normal overall morphology and maintained fully assembled tight junctions, the paracellular permeability to 4-kDa dextran, but not the paracellular permeability to 500-kDa dextran, was greatly increased. This report reveals for the first time that a pathogen can regulate the size-selective paracellular permeability of epithelial cells in order to elicit a disease process.

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High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02187-15 ◽

2015 ◽

Vol 81 (22) ◽

pp. 7881-7887 ◽

Cited By ~ 2

Author(s):

Takanori Kumagai ◽

Tomoki Ozawa ◽

Momoko Tanimoto ◽

Masafumi Noda ◽

Yasuyuki Matoba ◽

...

Keyword(s):

Escherichia Coli ◽

Host Cells ◽

Resting Cells ◽

Knockout Mutant ◽

Double Knockout ◽

Biosynthetic Genes ◽

T7 Promoter ◽

Α Subunit ◽

Content Type ◽

E Coli

ABSTRACTPreviously, we successfully cloned ad-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsAtodcsJ) fromd-CS-producingStreptomyces lavendulaeATCC 11924. In this study, we put fourd-CS biosynthetic genes (dcsC,dcsD,dcsE, anddcsG) in tandem under the control of the T7 promoter in anEscherichia colihost. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. Whenl-serine and hydroxyurea (HU), the precursors ofd-CS, were incubated together with theE. coliresting cell suspension, the cells produced significant amounts ofd-CS (350 ± 20 μM). To increase the productivity ofd-CS, thedcsJgene, which might be responsible for thed-CS excretion, was connected downstream of the four genes. TheE. coliresting cells harboring the five genes producedd-CS at 660 ± 31 μM. ThedcsDgene product, DcsD, formsO-ureido-l-serine fromO-acetyl-l-serine (OAS) and HU, which are intermediates ind-CS biosynthesis. DcsD also catalyzes the formation ofl-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, theE. colichromosomalcysJandcysKgenes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the fourd-CS biosynthetic genes, together withdcsJ, were incubated withl-serine and HU, thed-CS production was 980 ± 57 μM, which is comparable to that ofd-CS-producingS. lavendulaeATCC 11924 (930 ± 36 μM).

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Secretin of the Enteropathogenic Escherichia coli Type III Secretion System Requires Components of the Type III Apparatus for Assembly and Localization

Infection and Immunity ◽

10.1128/iai.71.6.3310-3319.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3310-3319 ◽

Cited By ~ 114

Author(s):

Annick Gauthier ◽

Jose Luis Puente ◽

B. Brett Finlay

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Biochemical Analysis ◽

Host Cells ◽

Type Iii Effectors ◽

Type Iii ◽

Secretion Pathway ◽

Detergent Extraction ◽

Inner Membrane Protein ◽

And Function

ABSTRACT At least 16 proteins are thought to be involved in forming the enteropathogenic Escherichia coli (EPEC) type III translocation apparatus which delivers virulence factors into host cells, yet their function and location have not been determined. A biochemical analysis was performed on three components: EscN, a predicted cytoplasmic ATPase; EscV, a predicted inner membrane protein; and EscC, a predicted outer membrane secretin. Wild-type EPEC and mutants constructed in these genes were fractionated by lysozyme treatment, ultracentrifugation, and selective detergent extraction. Fractionation revealed that the type III effectors Tir and EspB required a complete type III apparatus for any degree of export by EPEC, suggesting a continuous channel. Epitope-tagged EscC, EscV, and EscN were localized by fractionation, confirming computer modeling predictions for their location. Transcomplementation experiments revealed that localization of EscV and EscN was unaffected by mutations in other examined type III components. Remarkably, localization of EscC was altered in escV or escN mutants, where EscC accumulated in the periplasm. EscC was correctly localized in the escF needle component mutant, indicating that secretin localization is independent of needle formation. These data indicate that, contrary to previous indications, correct insertion and function of EscC secretin in the outer membrane depends not only on the sec-dependent secretion pathway but also on other type III apparatus components.

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The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT

Infection and Immunity ◽

10.1128/iai.00412-19 ◽

2019 ◽

Vol 87 (10) ◽

Cited By ~ 1

Author(s):

Tracy H. Hazen ◽

David A. Rasko

Keyword(s):

Escherichia Coli ◽

Complete Genome ◽

Genomic Analysis ◽

Host Cells ◽

Comparative Genomic ◽

Content Type ◽

E Coli ◽

Type Iii ◽

Severe Diarrhea ◽

Typical Epec

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.

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Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽

10.1099/mic.0.28368-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 143-152 ◽

Cited By ~ 12

Author(s):

Ciara M. Shaver ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Disease Severity ◽

Type Iii Secretion ◽

Disease Process ◽

Effector Proteins ◽

Host Cells ◽

Secreted Proteins ◽

Secretion Systems ◽

Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

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EspZ of Enteropathogenic and Enterohemorrhagic Escherichia coli Regulates Type III Secretion System Protein Translocation

mBio ◽

10.1128/mbio.00317-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 29

Author(s):

Cedric N. Berger ◽

Valerie F. Crepin ◽

Kobi Baruch ◽

Aurelie Mousnier ◽

Ilan Rosenshine ◽

...

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Protein Translocation ◽

Type Iii Secretion System ◽

Secretion System ◽

Enterohemorrhagic Escherichia Coli ◽

Host Cells ◽

Dependent Manner ◽

Content Type ◽

Type Iii

ABSTRACTTranslocation of effector proteins via a type III secretion system (T3SS) is a widespread infection strategy among Gram-negative bacterial pathogens. Each pathogen translocates a particular set of effectors that subvert cell signaling in a way that suits its particular infection cycle. However, as effector unbalance might lead to cytotoxicity, the pathogens must employ mechanisms that regulate the intracellular effector concentration. We present evidence that the effector EspZ controls T3SS effector translocation from enteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coli. Consistently, an EPECespZmutant is highly cytotoxic. Following ectopic expression, we found that EspZ inhibited the formation of actin pedestals as it blocked the translocation of Tir, as well as other effectors, including Map and EspF. Moreover, during infection EspZ inhibited effector translocation following superinfection. Importantly, while EspZ of EHEC O157:H7 had a universal “translocation stop” activity, EspZ of EPEC inhibited effector translocation from typical EPEC strains but not from EHEC O157:H7 or its progenitor, atypical EPEC O55:H7. We found that the N and C termini of EspZ, which contains two transmembrane domains, face the cytosolic leaflet of the plasma membrane at the site of bacterial attachment, while the extracellular loop of EspZ is responsible for its strain-specific activity. These results show that EPEC and EHEC acquired a sophisticated mechanism to regulate the effector translocation.IMPORTANCEEnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coli(EHEC) are important diarrheal pathogens responsible for significant morbidity and mortality in developing countries and the developed world, respectively. The virulence strategy of EPEC and EHEC revolves around a conserved type III secretion system (T3SS), which translocates bacterial proteins known as effectors directly into host cells. Previous studies have shown that when cells are infected in two waves with EPEC, the first wave inhibits effector translocation by the second wave in a T3SS-dependent manner, although the factor involved was not known. Importantly, we identified EspZ as the effector responsible for blocking protein translocation following a secondary EPEC infection. Interestingly, we found that while EspZ of EHEC can block protein translocation from both EPEC and EHEC strains, EPEC EspZ cannot block translocation from EHEC. These studies show that EPEC and EHEC employ a novel infection strategy to regulate T3SS translocation.

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Characterization of the hrpF Pathogenicity Peninsula of Xanthomonas oryzae pv. oryzae

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0546 ◽

2005 ◽

Vol 18 (6) ◽

pp. 546-554 ◽

Cited By ~ 46

Author(s):

Akiko Sugio ◽

Bing Yang ◽

Frank F. White

Keyword(s):

Gene Clusters ◽

Hypersensitive Reaction ◽

Xanthomonas Oryzae ◽

Host Cells ◽

Host Specialization ◽

Rice Cultivars ◽

Type Iii Effectors ◽

Type Iii ◽

The Right ◽

And Function

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpa1, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.

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Dynamics of the Type III Secretion System Activity of Enteropathogenic Escherichia coli

mBio ◽

10.1128/mbio.00303-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 37

Author(s):

Erez Mills ◽

Kobi Baruch ◽

Gili Aviv ◽

Mor Nitzan ◽

Ilan Rosenshine

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Public Health Problem ◽

Effector Proteins ◽

Host Cells ◽

Global Public Health ◽

Secretion Systems ◽

Comprehensive Description ◽

Content Type ◽

Type Iii

ABSTRACT Type III secretion systems (TTSSs) are employed by pathogens to translocate host cells with effector proteins, which are crucial for virulence. The dynamics of effector translocation, behavior of the translocating bacteria, translocation temporal order, and relative amounts of each of the translocated effectors are all poorly characterized. To address these issues, we developed a microscopy-based assay that tracks effector translocation. We used this assay alongside a previously described real-time population-based translocation assay, focusing mainly on enteropathogenic Escherichia coli (EPEC) and partly comparing it to Salmonella. We found that the two pathogens exhibit different translocation behaviors: in EPEC, a subpopulation that formed microcolonies carried out most of the translocation activity, while Salmonella executed protein translocation as planktonic bacteria. We also noted variability in host cell susceptibility, with some cells highly resistant to translocation. We next extended the study to determine the translocation dynamics of twenty EPEC effectors and found that all exhibited distinct levels of translocation efficiency. Further, we mapped the global effects of key TTSS-related components on TTSS activity. Our results provide a comprehensive description of the dynamics of the TTSS activity of EPEC and new insights into the mechanisms that control the dynamics. IMPORTANCE EPEC and the closely related enterohemorrhagic Escherichia coli (EHEC) represent a global public health problem. New strategies to combat EPEC and EHEC infections are needed, and development of such strategies requires better understanding of their virulence machinery. The TTSS is a critical virulence mechanism employed by these pathogens, and by others, including Salmonella. In this study, we aimed at elucidating new aspects of TTSS function. The results obtained provide a comprehensive description of the dynamics of TTSS activity of EPEC and new insights into the mechanisms that control these changes. This knowledge sets the stage for further analysis of the system and may accelerate the development of new ways to treat EPEC and EHEC infections. Further, the newly described microscopy-based assay can be readily adapted to study the dynamics of TTSS activity in other pathogens.

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SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System

Infection and Immunity ◽

10.1128/iai.73.7.4327-4337.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4327-4337 ◽

Cited By ~ 50

Author(s):

Kristen J. Kanack ◽

J. Adam Crawford ◽

Ichiro Tatsuno ◽

Mohamed A. Karmali ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Hela Cells ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Bacterial Attachment ◽

Host Cells ◽

Dependent Manner ◽

Cell Infection ◽

Type Iii

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

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EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli

Biochemical Journal ◽

10.1042/bj20111620 ◽

2012 ◽

Vol 442 (1) ◽

pp. 119-125 ◽

Cited By ~ 28

Author(s):

Neta Sal-Man ◽

Wanyin Deng ◽

B. Brett Finlay

Keyword(s):

Escherichia Coli ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Integral Membrane Protein ◽

Secretion System ◽

Host Cells ◽

Enteropathogenic Escherichia Coli ◽

Bacterial Membranes ◽

Type Iii ◽

Protein Functions

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.

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