scholarly journals Functional analysis of EspB from enterohaemorrhagic Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3277-3286 ◽  
Author(s):  
Hao-Jie Chiu ◽  
Wan-Jr Syu
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
The Other ◽  
Host Cells ◽  
Type Iii ◽  
Enterohaemorrhagic Escherichia Coli

In enterohaemorrhagic Escherichia coli (EHEC), the type III secretion protein EspB is translocated into the host cells and plays an important role in adherence, pore formation and effector translocation during infection. The secretion domain of EspB has been mapped previously. To define the other functional determinants of EspB, several plasmids encoding different fragments of EspB were created and analysed to see which of them lost the functions of the full-length molecule. One finding was that residues 118–190 of EspB were required for both efficient translocation of EspB and interaction of EspB with EspA. Additionally, the segment consisting of residues 217–312 was necessary for bacterial adherence. Furthermore, a predicted transmembrane domain (residues 99–118) was found to be critical for EHEC to cause red blood cell haemolysis, presumably by forming pores in the cell membrane. The same segment was also important for actin accumulation induced beneath the bacterial-attachment site. Taken together, these data indicate that the EspB protein (312 residues in total) has functions associated with its different regions. These regions may interact with each other or with other components of the type III system to orchestrate the intricate actions of EHEC during infection.

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

2005 ◽  
Vol 73 (7) ◽  
pp. 4327-4337 ◽  
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.

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 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.

Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157

2003 ◽  
Vol 31 (1) ◽  
pp. 98-103 ◽  
Author(s):  
A.J. Roe ◽  
D.E.E. Hoey ◽  
D.L. Gally
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Gastrointestinal Disease ◽  
Secretion System ◽  
Effector Proteins ◽  
Intimate Contact ◽  
Ongoing Research ◽  
Type Iii ◽  
Enterohaemorrhagic Escherichia Coli

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 causes gastrointestinal disease with the potential for life-threatening sequelae. Although Shiga-like toxins are responsible for much of the serious pathology in humans, the bacterium also possesses a type III protein secretion system that is responsible for intimate attachment to host intestinal mucosa. This sophisticated interaction requires co-ordination that is governed by environmental and genetic factors. Ongoing research supports the following model for how EHEC enables and controls this process: (i) specific environmental cues that are present in the host result in the expression of a number of adhesins, including fimbriae, which allow the initial binding to the mucosal surface. The same conditions support the expression of the basal type III secretion apparatus; (ii) targeting and assembly of the translocon requires both an mRNA signal and chaperones, with coupled translation and secretion of translocon proteins, EspA, B and D; (iii) opening up of a conduit between the bacterium and host cell releases a cytoplasmic pool of effector proteins. A consequence of this is increased expression of particular effector proteins. Potentially, different proteins could be released into the cell at different times or have activities modulated with time; (iv) intimate contact between the translocated intimin receptor (Tir) and the bacterial surface factor intimin requires translocon expression to be down-regulated and translocon filaments to be lost. Fluorescent protein fusions allow contact-mediated regulation and protein targeting through the type III secretion system to be studied in detail.

scholarly journals Recruitment and membrane interactions of host cell proteins during attachment of enteropathogenic and enterohaemorrhagic Escherichia coli

2012 ◽  
Vol 445 (3) ◽  
pp. 383-392 ◽  
Author(s):  
Diana Munera ◽  
Eric Martinez ◽  
Svetlana Varyukhina ◽  
Arvind Mahajan ◽  
Jesus Ayala-Sanmartin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Host Cell Proteins ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Attachment Sites ◽  
Infected Cells

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins NHERF2 (Na+/H+ exchanger regulatory factor 2) and AnxA2 (annexin A2), which are involved in regulation of intestinal ion channels, are recruited to the bacterial attachment sites. Using a stable HeLa-NHERF2 cell line, we found partial co-localization of AnxA2 and NHERF2; in EPEC-infected cells, AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerizes and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial effector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.

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

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
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.

Controlling injection: regulation of type III secretion in enterohaemorrhagic Escherichia coli

2009 ◽  
Vol 17 (8) ◽  
pp. 361-370 ◽  
Author(s):  
Jai J. Tree ◽  
Eliza B. Wolfson ◽  
Dai Wang ◽  
Andrew J. Roe ◽  
David L. Gally
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Type Iii ◽  
Enterohaemorrhagic Escherichia Coli

scholarly journals Posttranscriptional Control of Microbe-Induced Rearrangement of Host Cell Actin

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Charley C. Gruber ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Bacterial Attachment ◽  
Posttranscriptional Control ◽  
Content Type ◽  
Type Iii ◽  
Pedestal Formation

ABSTRACTRemodeling of the host cytoskeleton is a common strategy employed by bacterial pathogens. Although there is vigorous investigation of the cell biology underlying these bacterially mediated cytoskeleton modifications, knowledge of the plasticity and dynamics of the bacterial signaling networks that regulate the expression of genes necessary for these phenotypes is lacking. EnterohemorrhagicEscherichia coliattaches to enterocytes, forming pedestal-like structures. Pedestal formation requires the expression of the locus-of-enterocyte-effacement (LEE) andespFugenes. The LEE encodes a molecular syringe, a type III secretion system (T3SS) used by pathogens to translocate effectors such as EspFu into the host cell. By using a combination of genetic, biochemical, and cell biology approaches, we show that pedestal formation relies on posttranscriptional regulation by two small RNAs (sRNAs), GlmY and GlmZ. The GlmY and GlmZ sRNAs are unique; they have extensive secondary structures and work in concert. Although these sRNAs may offer unique insights into RNA and posttranscriptional biology, thus far, only one target and one mechanism of action (exposure of the ribosome binding site from theglmSgene to promote its translation) has been described. Here we uncovered new targets and two different molecular mechanisms of action of these sRNAs. In the case of EspFu expression, they promote translation by cleavage of the transcript, while in regard to the LEE, they promote destabilization of the mRNA. Our findings reveal that two unique sRNAs act in concert through different molecular mechanisms to coordinate bacterial attachment to mammalian cells.IMPORTANCEPathogens evolve by horizontal acquisition of pathogenicity islands. We describe here how two sRNAs, GlmY and GlmZ, involved in cellular metabolism and cellular architecture, through the posttranscriptional control of GlmS (the previously only known target of GlmY and GlmZ), which controls amino sugar synthesis, have been coopted to modulate the expression of virulence. These sRNAs quickly allow for plasticity in gene expression in order for enterohemorrhagicEscherichia colito fine-tune the expression of its complex type III secretion machinery and its effectors to promote bacterial attachment and subsequent actin rearrangement on host cells. Pedestal formation is a very dynamic process. Many of the genes necessary for pedestal formation are located within the same operon to evolutionarily guarantee that they are inherited together. However, it is worth noting that within these operons, several genes need to yield more proteins than others and that these differences cannot be efficiently regulated at the transcriptional level.

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

2012 ◽  
Vol 442 (1) ◽  
pp. 119-125 ◽  
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.

scholarly journals Efa-1/LifA mediates intestinal colonization of calves by enterohaemorrhagic Escherichia coli O26 : H– in a manner independent of glycosyltransferase and cysteine protease motifs or effects on type III secretion

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2527-2536 ◽  
Author(s):  
Victoria Deacon ◽  
Francis Dziva ◽  
Pauline M. van Diemen ◽  
Gad Frankel ◽  
Mark P. Stevens
Keyword(s):  
Escherichia Coli ◽  
Cysteine Protease ◽  
Type Iii Secretion ◽  
Intestinal Colonization ◽  
Fluid Accumulation ◽  
Faecal Excretion ◽  
E Coli ◽  
Type Iii ◽  
Enterohaemorrhagic Escherichia Coli

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of animal and zoonotic pathogens of worldwide importance. Our previous research established that intestinal colonization of calves by EHEC serotypes O5 : H– and O111 : H– requires EHEC factor for adherence (Efa-1), also known as lymphostatin (LifA). Towards an understanding of the mode of action of Efa-1/LifA, chromosomal in-frame deletions of predicted glycosyltransferase (DXD) and cysteine protease (CHD) motifs were created in a Δstx1 derivative of EHEC O26 : H–. The magnitude and duration of faecal excretion of EHEC O26 : H– were significantly reduced by null mutation of efa-1/lifA, but were not impaired by ΔDXD or ΔCHD mutations, in contrast to observations made with truncated Efa-1/LifA mutants of Citrobacter rodentium in mice. Although C. rodentium Efa-1/LifA influences the induction of colonic hyperplasia in mice, EHEC O26 : H– Efa-1/LifA was not required for fluid accumulation or neutrophil recruitment in bovine ileal loops. In contrast to observations with EHEC O5 : H– or O111 : H– mutants, inactivation of efa-1/lifA in EHEC O26 : H– did not significantly affect adherence or secretion of type III secreted proteins that play pivotal roles in calf colonization. Lymphostatin activity could not be reliably demonstrated in lysates of EHEC O26 : H–; however, deletion of the glycosyltransferase and cysteine protease motifs in Efa-1/LifA from enteropathogenic E. coli O127 : H6 abolished lymphostatin activity. Our data uncouple the role of Efa-1/LifA in calf colonization from effects on type III secretion and reinforce the potential for pathotype- and serotype-specific phenotypes.

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