Biomedical subjects

James B Kaper

Publications and source records attributed to James B Kaper.

At least 37 records · Page 2Linked to original sources

PubMed2004

Induction of interleukin-8 in T84 cells by Vibrio cholerae.

The induction of interleukin-8 (IL-8) in vitro has been suggested to correlate with the reactogenicity of Vibrio cholerae vaccine candidates. V. cholerae vaccine candidate 638, a hemagglutinin protease/hap-defective strain, was recently reported to be well tolerated in human volunteers, suggesting a role for Hap in reactogenicity. We examined the role of hap in the induction of IL-8 in intestinal epithelial T84 cells. Wild-type V. cholerae strains 3038 and C7258 and a vaccine candidate strain, JBK70, induced levels of IL-8 similar to those of their isogenic hap mutants. Supernatant containing Hap did not stimulate IL-8 production at a variety of concentrations tested, suggesting that Hap itself does not induce IL-8 production. Furthermore, supernatant from CVD115, which had deletions of hap and rtxA (encoding repeats in toxin) and was derived from a reactogenic strain, CVD110, induced IL-8 production in T84 cells in a dose-dependent manner. The IL-8-stimulating activity of CVD115 culture supernatants was growth phase dependent and was strongest in stationary phase cultures. This IL-8 stimulator(s) was resistant to heat treatment but sensitive to proteinase. Protease activity in vitro did not correlate with the reactogenicity of V. cholerae vaccine candidates. Our data suggest that Hap is not an IL-8 inducer in T84 cells and that the IL-8 stimulator in the supernatant of V. cholerae culture may play a role in reactogenicity.

Cholera Vaccines↗

PubMed2004

Long polar fimbriae contribute to colonization by Escherichia coli O157:H7 in vivo.

The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain.

Animals↗

PubMed2004

Evidence for a hybrid genomic island in verocytotoxin-producing Escherichia coli CL3 (serotype O113:H21) containing segments of EDL933 (serotype O157:H7) O islands 122 and 48.

Genomic O island 122 (OI-122) of the verocytotoxin-producing Escherichia coli (VTEC) strain EDL933 contains four putative virulence genes, Z4321, Z4326, Z4332, and Z4333. However, strain CL3 (serotype O113:H21) contains only Z4321, not the other three genes. To determine whether Z4321 is part of a different genomic island in CL3, a region of 27,293 bp up- and downstream of Z4321 was sequenced and found to contain elements of two different EDL933 genomic islands (OI-48 and OI-122) and a Yersinia pestis-like hemolysin/adhesin gene cluster. The region contained OI-48 genes Z1635, Z1636, and Z1637 at the left terminus and Z1641, Z1642, Z1643, and Z1644 at the right. The middle portion consisted of OI-48 gene Z1640, which was separated into three fragments by genomic segments including the Y. pestis cluster and EDL933 OI-122 genes Z4322, Z4321, and Z4318. In a PCR investigation of 36 VTEC strains of different serotypes, intact Z1640 was present in strains of serotypes O157:H7, O26:H11, O103:H2, O111:NM, and O145:NM, which are associated with hemolytic uremic syndrome and outbreaks. In contrast, fragmented Z1640 was seen in strains of nonepidemic serotypes, such as O91:H21 and O113:H21, and in animal serotypes that have not been associated with human disease, indicating that Z1640 might be a virulence gene.

Adhesins, Escherichia coli↗

PubMed2004

Transcriptional responses of intestinal epithelial cells to infection with Vibrio cholerae.

Vibrio cholerae is a noninvasive enteric bacterium that causes the severe diarrheal disease cholera. Candidate cholera vaccines have been engineered by deleting genes encoding known virulence factors in V. cholerae; however, many of these attenuated strains were still reactogenic in human volunteers. In this study, DNA arrays were utilized to monitor the transcriptional responses of human intestinal epithelial cells (T84) to eight strains of V. cholerae, including attenuated, toxigenic, and environmental isolates. cDNA probes generated from host RNA samples were hybridized against low- and high-density gene arrays. V. cholerae induced the transcription of a variety of host genes and repressed the expression of a lower number of genes. Expression patterns were confirmed for certain genes by reverse transcriptase PCR and enzyme-linked immunosorbent assays. A core subset of genes was found to be differentially regulated in all experiments. These genes included genes involved in innate mucosal immunity, intracellular signaling, and cellular proliferation. Reactogenic vaccine strains induced greater expression of genes for certain proinflammatory cytokines than nonreactogenic strains. Wild-type and attenuated derivatives induced and repressed many genes in common, although there were differences in the transcription profiles. These results indicate that the types of host genes modulated by attenuated V. cholerae, and the extent of their induction, may mediate the symptoms seen with reactogenic cholera vaccine strains.

Chemokines↗

PubMed2003

Age-specific frequencies of antibodies to Escherichia coli verocytotoxins (Shiga toxins) 1 and 2 among urban and rural populations in southern Ontario.

In 173 urban residents and 232 rural dairy-farm residents (age range, 0-70 years) who were stratified for age, the frequency of antiverocytotoxin 2 antibodies (VT2 Abs) (frequency in urban residents, 46%; frequency in rural residents, 65%) was significantly higher than that of antiverocytotoxin 1 antibodies (VT1 Abs) (frequency in urban residents, 12%; frequency in rural residents, 39%) (P< or =.001). The frequency of VT2 Abs (93%) was also significantly higher than that of VT1 Abs (50%) in 14 patients with hemolytic uremic syndrome (HUS) associated with verocytotoxin-producing Escherichia coli (VTEC) strains that expressed both toxins. In urban residents, the frequency of both antibodies tended to decrease between the first and the second decades of life, and it then increased until the fifth decade of life, before, in the case of VT2 Abs, decreasing again. This pattern, which inversely reflects the age-related incidence of HUS, is consistent with a role for antiverocytotoxin antibodies in protective immunity. In dairy-farm residents, peak frequencies of antibodies to both toxins occurred during the first decade of life and remained elevated for 3 decades before decreasing, a pattern consistent with frequent exposure to bovine VTEC from an early age.

Adolescent↗

PubMed2003

Bacteria-host communication: the language of hormones.

The interbacterial communication system known as quorum sensing (QS) utilizes hormone-like compounds referred to as autoinducers to regulate bacterial gene expression. Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is the agent responsible for outbreaks of bloody diarrhea in several countries. We previously proposed that EHEC uses a QS regulatory system to "sense" that it is within the intestine and activate genes essential for intestinal colonization. The QS system used by EHEC is the LuxS/autoinducer 2 (AI-2) system extensively involved in interspecies communication. The autoinducer AI-2 is a furanosyl borate diester whose synthesis depends on the enzyme LuxS. Here we show that an EHEC luxS mutant, unable to produce the bacterial autoinducer, still responds to a eukaryotic cell signal to activate expression of its virulence genes. We have identified this signal as the hormone epinephrine and show that beta- and alpha-adrenergic antagonists can block the bacterial response to this hormone. Furthermore, using purified and in vitro synthesized AI-2 we showed that AI-2 is not the autoinducer involved in the bacterial signaling. EHEC produces another, previously undescribed autoinducer (AI-3) whose synthesis depends on the presence of LuxS. These results imply a potential cross-communication between the luxS/AI-3 bacterial QS system and the epinephrine host signaling system. Given that eukaryotic cell-to-cell signaling typically occurs through hormones, and that bacterial cell-to-cell signaling occurs through QS, we speculate that QS might be a "language" by which bacteria and host cells communicate.

Bacterial Proteins↗

PubMed2003

Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells.

The type III secretion system (TTSS) of enteropathogenic Escherichia coli (EPEC) has been associated with the ability of these bacteria to induce secretion of proinflammatory cytokines, including interleukin-8 (IL-8), in cultured epithelial cells. However, the identity of the effector molecule directly involved in this event is unknown. In this study, we determined that the native flagellar filament and its flagellin monomer are activators of IL-8 release in T84 epithelial cells. Supernatants of wild-type EPEC strain E2348/69 and its isogenic mutants deficient in TTSS (escN) and in production of intimin (eae), grown in Luria-Bertani broth, elicited similar amounts of IL-8 secretion by T84 cells. In contrast, supernatants of EPEC fliC mutants and of B171, a nonflagellated EPEC strain, were defective in inducing IL-8 release, a phenotype that was largely restored by complementation of the fliC gene in the mutant lacking flagella. Purified flagella from E. coli K-12, EPEC serotypes H6 and H34, and enterohemorrhagic E. coli serotype H7 all induced IL-8 release in T84 cells. Induction of IL-8 by purified flagella or His-tagged FliC from EPEC strain E2348/69 was dose dependent and was blocked by a polyclonal anti-H6 antibody. Finally, the mitogen-activated protein kinases (Erk1 and -2 and Jnk) were phosphorylated in flagellin-treated T84 cells, and inhibition of the p38 and Erk pathways significantly decreased the IL-8 response induced by EPEC flagellin. Our data clearly indicate that FliC of EPEC is sufficient to induce IL-8 release in T84 cells and that activation of the Erk and p38 pathways is required for IL-8 induction.

Adhesins, Bacterial↗

PubMed2003

Multiple elements controlling adherence of enterohemorrhagic Escherichia coli O157:H7 to HeLa cells.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

Bacterial Adhesion↗

PubMed2003

Etiology of acute diarrhea in adults in southwestern Nigeria.

Stool specimens from 113 adult outpatients with diarrhea in southwestern Nigeria and 63 controls were examined for bacterial and parasitic enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) (P < 0.02), enteroaggregative E. coli (EAEC) (P < 0.02), and Entamoeba histolytica (P < 0.0002) were significantly associated with diarrhea. Salmonella, Shigella, nontoxigenic Vibrio cholerae, other categories of diarrheagenic E. coli, as well as a variety of helminths were recovered more frequently from the stools of patients than from the stools of controls but did not show a significant association with disease. Multiple pathogens were recovered from 36.3% of specimens, and bloody diarrhea was commonly associated with E. histolytica and diarrheagenic E. coli infections. The majority of EHEC isolates were non-O157 strains that carried the stx(2) gene. Of the 23 EHEC-infected patients, 12 (52.2%) presented during the 10th week of the study. EHEC strains isolated within this cluster were more likely to hybridize with the enterohemolysin gene probe, to be nonmotile and sorbitol positive, and to fail to agglutinate O157 antisera. Pulsed-field gel electrophoresis demonstrated that the only strains with XbaI profiles that occurred more than once were isolated during the 10th and 11th weeks of the study, suggesting an outbreak. The study has demonstrated that E. histolytica, EHEC, and EAEC are important diarrheal pathogens within the study area and that sporadic and epidemic EHEC infections occur in developing as well as developed countries. Routine surveillance for diarrheagenic E. coli, even only at the tertiary-care level, would be useful in identifying outbreaks and assist in identifying environmental reservoirs and transmission routes.

Acute Disease↗

PubMed2003

Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease.

The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five "seropathotypes" (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an "incomplete" OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001).

Animals↗

PubMed2003

Fitness cost of the green fluorescent protein in gastrointestinal bacteria.

There are surprisingly few studies that have successfully used the green fluorescent protein (GFP) as a quantitative reporter in selection experiments screening for inducible bacterial promoters. One explanation is that GFP expression may confer a fitness cost for bacteria. To test this possibility, we monitored the doubling time in enteric bacteria expressing GFP. Four bacterial species, Escherichia coli, enterohaemorrhagic E. coli, Shigella flexneri, Salmonella typhi, and Vibrio cholerae, were examined. The level of GFP expression was varied by using a salt-inducible promoter. After accounting for the increase in doubling time resulting from elevated osmolarity, the doubling time of all bacteria was found to increase proportionally with GFP expression, and some strains were more affected than others. Cultures of the bacteria most affected by GFP exhibited a proportion of elongated cells, which suggests that GFP production could interfere with cell division in these strains. The results in this study show that GFP is costly to bacteria and suggest that overly active promoters should be difficult to obtain from a genomic promoter library. They also suggest that the chances of succeeding in using GFP as a reporter in selection experiments are increased by growing the bacteria for the fewest number of generations and by subduing the expression of GFP whenever possible, such as by using a low copy vector to clone the library.

Bacteria↗

PubMed2002

Antibodies to intimin and Escherichia coli secreted proteins A and B in patients with enterohemorrhagic Escherichia coli infections.

Enterohemorrhagic Escherichia coli produce an attaching and effacing lesion upon adhering to the intestinal epithelium. Bacterial factors involved in this histopathology include the intimin adhesin and E. coli secreted proteins (Esps) A and B. In this study we investigated the serum antibody responses to recombinant E. coli O157:H7 intimin, EspA, and EspB by immunoblotting. Canadian patients with O157:H7 infection (n=10), Swedish patients with O157:H7 (n=21), non-O157 (n=18), or infection from which the serotype was not available (n=3), and asymptomatic household members (n=25) were studied and compared with Canadian (n=20) and Swedish controls (n=52). In Canadian patients, IgG antibodies to intimin, EspA, and EspB were analyzed, in Swedish patients and their household members IgA, IgG, and IgM antibodies to EspA and EspB were studied. Patients and household members mounted an antibody response to the antigens. Significantly more patients developed an acute response to EspB compared with controls (P<0.01 Canadian patients, P<0.0001 Swedish patients). EspB IgA, IgG, and IgM had a specificity of 100%, 86%, and 86%, positive predictive value of 100%, 83%, and 81%, and sensitivity of 57%, 69%, and 63%, respectively, and appear to be an appropriate assay for the detection of EHEC infection. In cases of hemolytic uremic syndrome or hemorrhagic colitis this assay may be useful when a fecal strain has not been isolated, or in epidemics of non-O157 infection.

Adhesins, Bacterial↗

PubMed2002

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions.

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.

Amino Acid Sequence↗

PubMed2002

Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli.

Quorum sensing is a cell-to-cell signalling mechanism in which bacteria secrete hormone-like compounds called autoinducers. When these auto-inducers reach a certain threshold concentration, they interact with bacterial transcriptional regulators, thereby regulating gene expression. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 as well as E. coli K-12 produces the autoinducer-2 (AI-2), which is synthesized by the product of the luxS gene, and previous work from our laboratory has shown that genes encoding the EHEC type III secretion system were activated by quorum sensing. Recently, by hybridizing an E. coli K-12 gene array with cDNA synthesized from RNA extracted from EHEC strain 86-24 and its isogenic luxS mutant, we observed that other potential virulence-associated factors, such as genes encoding the expression and assembly of flagella, motility and chemotaxis, were also activated by quorum sensing. The array data also indicated that several genes encoding putative E. coli regulators were controlled by quorum sensing. In this report, we describe a two-component system regulated by quorum sensing that shares homology with Salmonella typhimurium PmrAB, which we have named quorum sensing E. coli regulator B and C (QseBC). The qseBC genes, previously identified only as open reading frames b3025 and b3026, are organized in an operon in the E. coli chromosome, with qseB encoding the response regulator and qseC the sensor kinase. We confirmed the regulation of qseBC by quorum sensing using qseB::lacZ transcriptional fusions and characterized the phenotypes of an isogenic qseC mutation in EHEC. This mutant expressed less flagellin and had reduced motility compared with the wild-type and complemented strains. Transcription of flhD, fliA, motA and fliC::lacZ fusions was decreased in the qseC mutant, suggesting that qseBC is a transcriptional regulator of flagella genes. A qseC mutant was also generated in E. coli K-12 strain MC1000 that showed the same phenotypes as the EHEC mutant, indicating that qseBC regulates flagella and motility by quorum sensing in both EHEC and K-12. QseBC activates transcription of flhDC, which is the master regulator for the flagella and motility genes and, in the absence of flhD, QseBC failed to activate the transcription of fliA. Motility of a luxS, but not of a qseC, mutant can be restored by providing AI-2 exogenously as preconditioned media, suggesting that the qseC mutant is unable to respond to AI-2. However, QseC has no effect on the expression of other quorum sensing-controlled genes such as those encoding for the type III secretion system. These data indicate that QseBC is one component of the quorum-sensing regulatory cascade in both EHEC and K-12 that is involved in the regulation of flagella and motility genes, but that additional regulators in this cascade remain to be characterized.

DNA-Binding Proteins↗

PubMed2002

ToxR interferes with CRP-dependent transcriptional activation of ompT in Vibrio cholerae.

In pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus. The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR. In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase. Deletion of the crp gene completely abolishes OmpT expression. Comparison of ompT transcription levels in the isogenic crp-, toxR- and crp-toxR- mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP-dependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase. To provide further insights into the molecular mecha-nism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression. Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase (RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from -310 of the ompT promoter via DNA looping.

Bacterial Proteins↗

PubMed2002

The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells.

Enteropathogenic Escherichia coli (EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here that fliC insertional mutants constructed in two wild-type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. An E. coli K-12 strain harbouring the EPEC H6 fliC gene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild-type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non-motile EPEC. Isogenic strains mutated in perA (a transcriptional activator), bfpA (a type IV pilin), luxS (a quorum-sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco's modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid-encoded regulator, quorum sensing and epithelial cells.

Bacterial Adhesion↗

PubMed2002

StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor.

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.

Blood Proteins↗

PubMed2002

Characterization of Cah, a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli.

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.

Amino Acid Sequence↗