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J B Kaper

Publications and source records attributed to J B Kaper.

At least 55 records · Page 3Linked to original sources
PubMed1998

EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.

Amino Acid Sequence↗
PubMed1998

Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae.

The membrane proteins ToxR and ToxS regulate a variety of genes associated with the virulence of Vibrio cholerae, the agent of human cholera. One of the ToxRS-regulated genes is the ompU gene, which encodes a porin that may also act as an adhesin. To begin to understand the mechanism of ompU transcription activation by ToxRS, we performed genetic and biochemical studies on the ompU promoter. Deletions with a 5' end-point at or downstream of -128, relative to the start site for transcription, did not direct expression of a lacZ reporter gene in wild-type V. cholerae, although the -128 promoter fragment did direct ToxRS-dependent reporter gene activity under conditions of ToxR overexpression in E. coli. Consistent with the activation data is that membranes containing ToxR and ToxS caused a gel electrophoretic mobility shift when mixed at low concentrations with deletion fragments whose end-point is at -211, but not with -128 or -68 fragments. ToxRS membranes did shift the -128 fragment when added at higher concentrations. DNase I footprinting analysis of ompU promoter DNA complexed with ToxRS membranes demonstrated protection of three sites: an upstream site ranging from -238 to -139, and two downstream sites ranging from -116 to -58 and -53 to -24. Within the DNA protected from DNase I digestion by ToxRS membranes, there are no elements bearing similarity to those identified previously within the promoters of two other ToxR-dependent genes, ctxA and toxT. We suggest a model for transcription activation that involves sequential ToxR-binding events to distinct regions in the ompU promoter.

Adhesins, Bacterial↗
PubMed1998

Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements.

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.

Animals↗
PubMed1998

Diarrheagenic Escherichia coli.

Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler's diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (entero-pathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens.

Diarrhea↗
PubMed1998

Enteropathogenic Escherichia coli virulence genes encoding secreted signalling proteins are essential for modulation of Caco-2 cell electrolyte transport.

The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhea remains uncertain. In vitro, EPEC stimulates a rapid increase in short-circuit current (Isc) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion. This study has examined the roles of specific EPEC virulence proteins in this Isc response. EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport.

Adhesins, Bacterial↗
PubMed1998

Investigation of the roles of toxin-coregulated pili and mannose-sensitive hemagglutinin pili in the pathogenesis of Vibrio cholerae O139 infection.

In this study, adult volunteers were fed tcpA and mshA deletion mutants of V. cholerae O139 strain CVD 112 to determine the role of toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) in intestinal colonization. Eight of 10 volunteers who received CVD 112 or CVD 112 delta mshA shed the vaccine strains in their stools; the geometric mean peak excretion for both groups was 1.4 x 10(5) CFU/g of stool. In contrast, only one of nine recipients of CVD 112 delta tcpA shed vibrios in his stool (P < 0.01); during the first 24 h after inoculation, 3 x 10(2) CFU/g was recovered from this volunteer. All recipients of CVD 112 and 8 (80%) of the recipients of CVD 112 delta mshA developed at least a fourfold rise in vibriocidal titer after immunization. In contrast, only one (11%) of the nine recipients of CVD 112 delta tcpA developed a fourfold rise in vibriocidal titer (P < 0.01). We conclude that TCP are an important colonization factor of V. cholerae O139 and probably of El Tor V. cholerae O1. In contrast, MSHA does not appear to promote intestinal colonization in humans.

Adult↗
PubMed1998

Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro.

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.

Adhesins, Bacterial↗
PubMed1998

Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7.

We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagic Escherichia coli O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis. The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector. Recent completion of the LEE sequence from enteropathogenic E. coli (EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island. We have identified a total of 54 open reading frames in the EDL933 LEE. Of these, 13 fall within a putative P4 family prophage designated 933L. The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates. The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene. The rate of divergence is heterogeneous along the locus. Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among E. coli strains. Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell. This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general.

Amino Acid Sequence↗
PubMed1998

Virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118, a major group of STEC pathogens in calves.

Shiga toxin-producing Escherichia coli (STEC) strains of serogroup 0118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 0118 (0118:H16 [n = 38] and 0118:H- [n = 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hlyEHEC (hly from enterohemorrhagic E. coli). The strains' adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%; P < 0.001]). In conclusion, the vast majority of the 0118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (stx, hlyEHEC, and LEE). This virulence potential and the high prevalence of STEC 0118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.

Actins↗
PubMed1997

Insertion site of the locus of enterocyte effacement in enteropathogenic and enterohemorrhagic Escherichia coli differs in relation to the clonal phylogeny of the strains.

The locus of enterocyte effacement pathogenicity island confers the attaching and effacing histopathology on epithelial cells infected with enteropathogenic and enterohemorrhagic Escherichia coli. We investigated the site of insertion of the locus of enterocyte effacement in E. coli strains in relation to their evolution based on conservation of housekeeping proteins in these strains. The results indicate that the insertion site of the locus of enterocyte effacement varies according to the evolutionary lineage, suggesting that it has inserted at multiple times and sites during the evolution of these pathogens.

Bacterial Adhesion↗
PubMed1997

Longus pilus of enterotoxigenic Escherichia coli and its relatedness to other type-4 pili--a minireview.

Longus is a long pilus produced by human enterotoxigenic Escherichia coli (ETEC) which shares significant structural and biochemical features with class-B type-4 pili. These pili include the toxin-coregulated pilus (TCP) of Vibrio cholerae, the bundle-forming pilus (BFP) of enteropathogenic E. coli and both longus and the colonization factor antigen III (CFA/III) of ETEC. These pili are produced under defined growth conditions indicating that they are under the control of different regulatory elements. While TCP is chromosomally encoded, the remaining pili are encoded on large virulence plasmids. Longus and CFA/III are closely related pili although certain DNA and protein differences also exist between them. This may account for the differences in the regulation, surface presentation, antigenicity, and prevalence of these two pilins among ETEC. Neighboring lngA, a second open reading frame termed lngB was found which encodes a protein with significant homology to proteins which are part of a type-II secretory system such as XcpV, OutC, and PulO of Pseudomonas aeruginosa, Erwinia chrysanthemi, and Klebsiella pneumoniae, respectively. This suggests that lngB may be an accessory gene involved in biogenesis of longus.

Amino Acid Sequence↗
PubMed1997

Role of type 1 fimbriae in EPEC infections.

Several fimbriae have been implicated as potentially important in EPEC adhesion and pathogenesis. EPEC strain E2348/69 produced only bundle forming pili and type 1 fimbriae, and did not produce other accesory adhesins identified in EPEC strain B171. Cloning and mutagenesis of these EPEC fim genes indicated that type 1 pili had no affect on levels or patterns of adhesion to cultured human cells.

Animals↗
PubMed1997

Cloning of the RDEC-1 locus of enterocyte effacement (LEE) and functional analysis of the phenotype on HEp-2 cells.

EPEC Escherichia coli are an important cause of epidemic diarrhea in infants. The disease is characterized by attaching and effacing (A/E) lesions where the bacteria attach intimately to the enterocyte surface resulting in localized destruction of microvilli. A 35-kb chromosomal locus termed LEE (locus of enterocyte effacement) in an EPEC strain (E2348/69) has recently been found and is thought to contain all the necessary genes for A/E lesions. RDEC-1 is a strain of E. coli that causes diarrhea in rabbits by a similar mechanism and serves as a model for human EPEC disease. We report 1) the cloning of the RDEC-1 LEE, 2) show that the RDEC-1 LEE is similar in size to the LEE in E2348/69, 3) the RDEC-1 LEE possesses all four regions of the E2348/69 LEE, 4) there are restriction site polymorphisms between the RDEC-1 LEE and that of E2348/69, and 5) the RDEC-1 LEE clone is functionally similar to E2348/69 in its fluorescent actin staining test and suggests that the LEE may be sufficient for the production of A/E lesions by these strains.

Bacterial Adhesion↗
PubMed1997

Genetics of virulence of enteropathogenic E. coli.

Enteropathogenic E. coli (EPEC) are a major cause of diarrhea in infants throughout the world. Although this pathogen was described 50 years ago, it is only recently that the pathogenic mechanisms employed by this organism have been elucidated. The characteristic histopathology induced by this organism, called "attaching and effacing", consists of intimate adherence of the bacterium to the epithelial cell with marked cytoskeletal changes including effacement of microvilli. A 35 kb region of chromosomal DNA, called the LEE for locus of enterocyte effacement has recently been described which contains all known genes necessary for production of this characteristic histopathology. Within this region is the eae gene encoding intimin, a 94 kDa OMP involved in intimate adherence. Also within this region are genes encoding proteins secreted extracellularly by EPEC (esp) and a type III secretion apparatus (sep) which shares homology with similar systems in Yersinia, Shigella, and Salmonella. Additional genes on a 60 MDa plasmid encode a type IV pilus (BFP) and a positive transcriptional activator (per) of multiple chromosomal and plasmid virulence genes.

Animals↗
PubMed1997

Interactions between enteropathogenic Escherichia coli and host epithelial cells.

The pathogenesis of enteropathogenic Escherichia coli (EPEC) infection is emerging as a paradigm for a multistage microorganism-host cell interaction. Both type IV fimbriae and a type III secretion apparatus play principal roles in interactions between the bacteria and host cells. Recent data suggest that bacteria-induced signal transduction activates the receptor that allows tenacious adherence of the bacteria to the host cell surface.

Bacterial Adhesion↗