Steve L. Moseley

Genetic and Phenotypic Analysis of Escherichia coli with Enteropathogenic Characteristics Isolated from Seattle Children

Coliform colonies from children whose stools were submitted for microbiologic analysis were studied prospectively to determine the frequency of shedding of enteropathogenic Escherichia coli (EPEC). In total, 2225 isolates from 445 patients were probed with eaeA (encoding intimin) and the EAF (EPEC adherence factor) probe, and adherence and actin-aggregating phenotypes were determined. Twenty-five patients (5.6%) shed non-O157:H7 eaeA+ EAF- E. coli. Of these 25 patients, isolates from 5 produced Shiga toxins and from 3 possessed bfpA (encoding the bundle-forming pilus) sequences. Non-O157:H7 eaeA+ E. coli from 21 (84%) of 25 patients adhered locally to and aggregated actin in HeLa cells. Four patients shed nonadherent EAF+ eaeA- E. coli. Non-O157:H7 eaeA+ and EAF- isolates belonged to diverse electrophoretic types and classical and nonclassical enteropathogenic serotypes. EPEC are relatively common in stools submitted for analysis in this North American pediatric hospital. Their etiologic role in childhood diarrhea warrants elucidation.

Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis.

Escherichia coli that express Dr fimbriae and related adhesins recognize the common receptor decay accelerating factor. E. coli strains that express adhesins of the Dr family were postulated to be associated with cystitis (30-50%), pregnancy-associated pyelonephritis (30%), and chronic diarrhea (50%). In this study, we investigated the hypothesis that E. coli renal interstitial binding mediated by the Dr adhesin may be important for the development of chronic pyelonephritis. An insertional dra mutant, E. coli DR14, of the clinical E. coli isolate IH11128 bearing Dr fimbriae, was constructed and used to characterize persistence of infection and interstitial tropism in an experimental model of ascending pyelonephritis. Quantitative cultures of kidney homogenates indicated that Dr hemagglutinin positive (Dr+) E. coli IH11128 established a 1-yr colonization of renal tissue. In the Dr hemagglutinin negative (Dr-) group, 50% of animals cleared infection within 20 wk and 100% between 32 to 52 wk. Dr+ E. coli colonized the renal interstitium. Significant histological changes corresponding to tubulointerstitial nephritis including interstitial inflammation, fibrosis, and tubular atrophy were found in the kidney tissue of the Dr+ but not the Dr- group. A substantial amount of fimbrial antigen was detected in the parenchymal regions affected by interstitial inflammation and fibrosis. The obtained results are consistent with the hypothesis that mutation within the dra region, affecting E. coli binding to tubular basement membranes, prevented renal interstitial tropism and the development of the changes characteristically seen in tubulointerstitial nephritis.

Recruitment of CD55 and CD66e Brush Border-Associated Glycosylphosphatidylinositol-Anchored Proteins by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli That Infect the Human Polarized Intestinal Caco-2/TC7 Cells

ABSTRACT The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins.

The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea‐associated Escherichia coli to decay‐accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family—AFAI, AFAIII, and F1845—to bind to type IV collagen, and demonstrated that the collagen‐binding phenotype was unique to the Dr haemagglutinin. We employed site‐directed mutagenesis to demonstrate the requirement of a negatively charged amino‐acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF‐binding capability. These results suggest the existence of a conformational receptor‐binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.

Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli

The transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea‐associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papi genes of the P fimbrial adhesin, respectively. The 5′ termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine‐responsive regulatory protein (Lrp)‐binding sites which contained differentially methylated GATC sequences, a cAMP‐CRP‐binding site, and an integration host factor (IHF)‐binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.

Shiga-like toxin-producing Escherichia coli in Seattle children: A prospective study

BACKGROUND The frequency with which stools contain Shiga-like toxin producing Escherichia coli not belonging to serotype O157:H7 is unknown in the United States. The aim of this study was to determine the frequency with which these E. coli are present in stools from children from Seattle submitted for bacteriologic analysis. METHODS 2225 coliform colonies from 445 stools submitted for bacterial culture from Seattle children were probed with the structural genes of Shiga-like toxins I and II in a 1-year prospective study. The adherence and actin aggregating characteristics of these E. coli were subsequently determined. RESULTS Five (1.1%) patients had non-O157:H7 Shiga-like toxin producing E. coli, a rate of isolation higher than Shigella or Yersinia (0.2% each) but lower than Campylobacter (2.5%), E. coli O157:H7 (2.9%), or Salmonella (3.4%). Only one of the five patients had bloody diarrhea. None developed hemolytic uremic syndrome. All strains adhered in a localized pattern to, and induced actin aggregation in, HeLa cells, and produced a toxin that was lethal to Vero cells. CONCLUSIONS Non-O157:H7 Shiga-like toxin producing E. coli are present in stools submitted for bacterial culture in a North American childhood population. Their role in childhood diarrhea warrants better definition.

HrpA, a DEAH‐box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli

Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co‐ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH‐box RNA helicase‐encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site‐directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.

Expression of putative virulence factors of Escherichia coli O157:H7 differs in bovine and human infections.

ABSTRACT Escherichia coli O157:H7 is a commensal organism in cattle, but it is a pathogen in humans. This differential expression of virulence suggests that specific virulence factors are regulated differently in human and bovine hosts. To test this hypothesis, relative real-time reverse transcription-PCR was used to relate the expression of several putative virulence genes (eae, espA, stx2, rfbE, ehxA, and iha) to that of the “housekeeping” gene gnd during natural human and experimental bovine infection with E. coli O157:H7. We examined these genes in fecal samples from eight humans and four calves. iha and espA were significantly more expressed in bovine infections. rfbE and ehxA appeared to be more highly expressed in human infections, though these differences did not achieve statistical significance. Our results support the hypothesis that some virulence-associated genes of O157:H7 are differentially expressed in a host-specific manner.

Interaction of Dr Adhesin with Collagen Type IV Is a Critical Step in Escherichia coli Renal Persistence

ABSTRACT The pathogenic mechanism of recurrent or chronic urinary tract infection is poorly understood. Escherichia coli cells bearing Dr fimbriae display unique tropism to the basement membrane (BM)-renal interstitium that enables the bacteria to cause chronic pyelonephritis in experimental mice. The renal receptors for Dr-fimbriated E. coli are type IV collagen and decay-accelerating factor (DAF). We hypothesized that type IV collagen receptor-mediated BM-interstitial tropism is essential for E. coli to cause chronic pyelonephritis. To test the role of the type IV collagen tropism of Dr-fimbriated E. coli in renal persistence, we constructed an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to DAF and examined its virulence in the mouse model. The collagen-binding mutant DrI113T was eliminated from the mouse renal tissues in 6 to 8 weeks, while the parent strain caused persistent renal infection that lasted at least 14 weeks (P ≤ 0.02). Transcomplementation with the intact Dr operon restored collagen-binding activity, BM-interstitial tropism, and the ability to cause persistent renal infection. We conclude that type IV collagen binding mediated by DraE adhesin is a critical step for the development of persistent renal infection in a murine model of E. coli pyelonephritis.

A subfamily of Dr adhesins of Escherichia coli bind independently to decay-accelerating factor and the N-domain of carcinoembryonic antigen

Escherichia coli expressing the Dr family of adhesins adheres to epithelial cells by binding to decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell surface proteins. The attachment of bacteria expressing Dr adhesins to DAF induces clustering of DAF around bacterial cells and also recruitment of CEA-related cell adhesion molecules. CEA, CEACAM1, and CEACAM6 have been shown to serve as receptors for some Dr adhesins (AfaE-I, AfaE-III, DraE, and DaaE). We demonstrate that AfaE-I, AfaE-V, DraE, and DaaE adhesins bind to the N-domain of CEA. To identify the residues involved in the N-CEA/DraE interaction, we performed SPR binding analyses of naturally occurring variants and a number of randomly generated mutants in DraE and N-CEA. Additionally, we used chemical shift mapping by NMR to determine the surface of DraE involved in N-CEA binding. These results show a distinct CEA binding site located primarily in the A, B, E, and D strands of the Dr adhesin. Interestingly, this site is located opposite to the β-sheet encompassing the previously determined binding site for DAF, which implies that the adhesin can bind simultaneously to both receptors on the epithelial cell surface. The recognition of CEACAMs from a highly diverse DrCEA subfamily of Dr adhesins indicates that interaction with these receptors plays an important role in niche adaptation of E. coli strains expressing Dr adhesins.