scholarly journals Distribution, Functional Expression, and Genetic Organization of Cif, a Phage-Encoded Type III-Secreted Effector from Enteropathogenic and Enterohemorrhagic Escherichia coli

2007 ◽  
Vol 190 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Estelle Loukiadis ◽  
Rika Nobe ◽  
Sylvia Herold ◽  
Clara Tramuta ◽  
Yoshitoshi Ogura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Expression ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
E Coli ◽  
Type Iii ◽  
Related Phenotype ◽  
Enterocyte Effacement ◽  
Lambdoid Prophage

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.

scholarly journals Pas, a Novel Protein Required for Protein Secretion and Attaching and Effacing Activities of Enterohemorrhagic Escherichia coli

1998 ◽  
Vol 180 (17) ◽  
pp. 4370-4379 ◽  
Author(s):  
Andreas U. Kresse ◽  
Kai Schulze ◽  
Christina Deibel ◽  
Frank Ebel ◽  
Manfred Rohde ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
Reading Frame ◽  
E Coli ◽  
Genes Encoding ◽  
Enterocyte Effacement ◽  
Putative Member

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, andespD genes on the complementary strand and which exhibits high homology to the genes spiB fromSalmonella, yscD from Yersinia, andpscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated pas (protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.

scholarly journals Comparative Analysis of Locus of Enterocyte Effacement Pathogenicity Islands of Atypical Enteropathogenic Escherichia coli

2004 ◽  
Vol 72 (11) ◽  
pp. 6722-6728 ◽  
Author(s):  
Julia F. Gärtner ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Functional Expression ◽  
Secretion System ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Pathogenicity Islands ◽  
E Coli ◽  
Enterocyte Effacement ◽  
Pedestal Formation

ABSTRACT The pathogenicity of enteropathogenic Escherichia coli (EPEC) is linked to the locus of enterocyte effacement, or LEE, encoding a type III secretion system (T3SS) that directly transfers bacterial effector proteins into eukaryotic cells. Atypical diffusely adhering EPEC (DA-EPEC) strains that harbor homologues of the LEE but lack the EPEC adherence factor plasmid have been increasingly associated with outbreaks of diarrhea. In this study, we have completely sequenced and functionally characterized LEE pathogenicity islands derived from the clinical DA-EPEC isolates 3431 (O8:H−) and 0181 (O119:H9:K61). LEE3431 and LEE0181 exhibit genetic organization analogous to that of the prototype LEEE2348/69. Genes constituting the T3SS apparatus are highly conserved. However, LEE-encoded effector proteins exhibit major differences. Transfer and functional expression of LEE0181 in an E. coli XL1 blue MR background demonstrated that LEE0181 contains all the information for signal transduction and pedestal formation.

scholarly journals Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter

2004 ◽  
Vol 186 (16) ◽  
pp. 5486-5495 ◽  
Author(s):  
Xavier Charpentier ◽  
Eric Oswald
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Host Cell ◽  
Effector Proteins ◽  
Host Cells ◽  
Reporter System ◽  
Type Iii ◽  
Cycle Arrest ◽  
Lambdoid Prophage

ABSTRACT Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 β-lactamase. Translocation was detected directly in living host cells by using the fluorescent β-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.

scholarly journals Differential Modulation by Ca2+ of Type III Secretion of Diffusely Adhering Enteropathogenic Escherichia coli

2003 ◽  
Vol 71 (4) ◽  
pp. 1725-1732 ◽  
Author(s):  
Tina Ide ◽  
Silke Michgehl ◽  
Sabine Knappstein ◽  
Gerhard Heusipp ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Host Cells ◽  
Chromosomal Locus ◽  
Type Iii ◽  
Calcium Chelation ◽  
Host Plasma Membrane ◽  
Enterocyte Effacement

ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca2+ is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca2+. While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca2+-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery.

scholarly journals Polymorphisms within EspA Filaments of Enteropathogenic and Enterohemorrhagic Escherichia coli

2003 ◽  
Vol 71 (4) ◽  
pp. 2262-2265 ◽  
Author(s):  
Bianca C. Neves ◽  
Robert K. Shaw ◽  
Gad Frankel ◽  
Stuart Knutton
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Secretion System ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
Secreted Protein ◽  
Epec Strain ◽  
Lesion Formation ◽  
Type Iii

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) possess a filamentous type III secretion system (TTSS) employed to deliver effector proteins into host cells. EspA is a type III secreted protein which forms the filamentous extension to the TTSS and which interacts with host cells during early stages of attaching and effacing (A/E) lesion formation. By immunofluorescence, a polyclonal antibody previously raised to EspA from EPEC strain E2348/69 (O127:H6) stained ∼12-nm-diameter EspA filaments produced by this strain but did not stain similar filaments produced by EHEC serotype O157:H7. Similarly, an antibody that we subsequently raised to EHEC strain 85-170 (O157:H7) EspA stained ∼12-nm-diameter EspA filaments produced by strain 85-170 but did not stain E2348/69 EspA filaments. Given such heterogeneity between EPEC and EHEC EspA filaments, we examined polymorphisms of functional EspA filaments among different EPEC and EHEC serotypes. With use of the EPEC EspA antiserum, EspA filaments were observed only with EPEC serotypes O127:H6 and O55:H6, serotypes which encode an identical EspA protein. When stained with the EHEC EspA antiserum, EspA filaments were detected only on EHEC strains belonging to serotype O157:H7; the EHEC antiserum did, however, stain EspA filaments produced by the closely related EPEC serotype O55:H7 but not filaments of any other EPEC serotype tested. Such polymorphisms among functional EspA filaments of EPEC and EHEC would be expected to have important implications for the development of broad-range EspA-based vaccines.

scholarly journals Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
Maarten F. de Jong ◽  
Neal M. Alto
Keyword(s):  
Escherichia Coli ◽  
Defense Mechanisms ◽  
Immune Defense ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
E Coli ◽  
Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

scholarly journals 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

2019 ◽  
Vol 87 (10) ◽  
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.

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

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
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.

scholarly journals The Locus of Enterocyte Effacement-Encoded Effector Proteins All Promote Enterohemorrhagic Escherichia coli Pathogenicity in Infant Rabbits

2005 ◽  
Vol 73 (3) ◽  
pp. 1466-1474 ◽  
Author(s):  
Jennifer M. Ritchie ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Effector Proteins ◽  
Enterohemorrhagic Escherichia Coli ◽  
Lesion Formation ◽  
Specific Effects ◽  
Genes Encoding ◽  
Organ Specific ◽  
Rabbit Intestine ◽  
Enterocyte Effacement

ABSTRACT The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (∼10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by ∼10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.

scholarly journals Quorum-Sensing Escherichia coli Regulator A: a Regulator of the LysR Family Involved in the Regulation of the Locus of Enterocyte Effacement Pathogenicity Island in Enterohemorrhagic E. coli

2002 ◽  
Vol 70 (6) ◽  
pp. 3085-3093 ◽  
Author(s):  
Vanessa Sperandio ◽  
Caiyi C. Li ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Iii Secretion ◽  
Pathogenicity Island ◽  
The Other ◽  
E Coli ◽  
Type Iii ◽  
Enterocyte Effacement ◽  
K 12 ◽  
Lysr Family

ABSTRACT The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). The LEE comprises 41 open reading frames organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins. The first gene of LEE1 encodes the Ler regulator, which activates all the other genes within the LEE. We previously reported that the LEE genes were activated by quorum sensing through Ler (V. Sperandio, J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 96:15196-15201, 1999). In this study we report that a putative regulator in the E. coli genome is itself activated by quorum sensing. This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and E. coli K-12; and shares homology with the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively. We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing E. coli regulator A (QseA). We observed that QseA activated transcription of ler and therefore of the other LEE genes. An EHEC qseA mutant had a striking reduction of type III secretion activity, which was complemented when qseA was provided in trans. Similar results were also observed with a qseA mutant of EPEC. The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing.

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