Novel Avian Pathogenic Escherichia coli Genes Responsible for Adhesion to Chicken and Human Cell Lines

Abstract

Avian pathogenic Escherichia coli (APEC) is a major bacterial pathogen of commercial poultry contributing to extensive economic losses and contamination of the food chain. One of the initial steps in bacterial infection and successful colonization of the host is adhesion to the host cells. A random transposon mutant library (n = 1,300) of APEC IMT 5155 was screened phenotypically for adhesion to chicken (CHIC-8E11) and human (LoVo) intestinal epithelial cell lines. The detection and quantification of adherent bacteria were performed by a modified APEC-specific antibody staining assay using fluorescence microscopy coupled to automated VideoScan technology. Eleven mutants were found to have significantly altered adhesion to the cell lines examined. Mutated genes in these 11 "adhesion-altered mutants" were identified by arbitrary PCR and DNA sequencing. The genes were amplified from wild-type APEC IMT 5155, cloned, and transformed into the respective adhesion-altered mutants, and complementation was determined in adhesion assays. Here, we report contributions of the fdtA, rluD, yjhB, ecpR, and fdeC genes of APEC in adhesion to chicken and human intestinal cell lines. Identification of the roles of these genes in APEC pathogenesis will contribute to prevention and control of APEC infections.IMPORTANCE Avian pathogenic E. coli is not only pathogenic for commercial poultry but can also cause foodborne infections in humans utilizing the same attachment and virulence mechanisms. Our aim was to identify genes of avian pathogenic E. coli involved in adhesion to chicken and human cells in order to understand the colonization and pathogenesis of these bacteria. In contrast to the recent studies based on genotypic and bioinformatics data, we have used a combination of phenotypic and genotypic approaches for identification of novel genes contributing to adhesion in chicken and human cell lines. Identification of adhesion factors remains important, as antibodies elicited against such factors have shown potential to block colonization and ultimately prevent disease as prophylactic vaccines. Therefore, the data will augment the understanding of disease pathogenesis and ultimately in designing strategies against the infections.

Keywords: VideoScan; adhesion genes; avian pathogenic E. coli; chicken cell lines.

FIG 1

Rapid screening of transposon mutants using O2-antibody staining method. (A) CHIC-8E11 cells as a negative control in the DAPI channel. (B) Wild-type APEC IMT 5155. (C) Transposon mutant with low adhesion. (D) Transposon mutant with high adhesion.

FIG 2

Adhesion of APEC wild-type strain IMT5155 (average value normalized to 100) and its selected transposon mutants to CHIC-8E11 (red bars) and LoVo (blue bars) intestinal cells. The data from the initial screening of the transposon library are shown as median values and median absolute deviation (MAD) of two separate experiments in triplicate.

FIG 3

Motility assay of APEC wild-type strain IMT5155 and selected transposon mutants. The data are shown as median values and MAD of three separate experiments. SAEC5148, nonmotile strain used as a negative control.

FIG 4

Adhesion of APEC wild-type strain IMT5155, selected transposon mutants, and transposon mutants transformed with either empty vector pACYC177 (Neg, green bar) or pACYC177 plasmids bearing wild-type versions of transposon mutated genes (Comp, red bars) to CHIC-8E11 and LoVo intestinal epithelial cells. The data shown are median values and MAD of three separate experiments in sextuplicates. Wells not incubated with bacteria were used as a negative control.

FIG 5

Prevalences of the selected genes in various E. coli pathotypes. Gene prevalence is shown for genes (on the x axis) in E. coli pathotypes (y axis). Grayscale indicates color intensity change with the prevalence of gene of interest. Each rectangle corresponds to the prevalence of a gene of interest in particular pathotype. Pathotypes used include uropathogenic E. coli (UPEC), human intestinal commensal (fecal) E. coli (HFEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), sepsis-associated human E. coli isolated from sepsis patients (SAEC), enteroaggregative E. coli (EAEC), avian pathogenic E. coli (APEC), chicken intestinal commensal (avian fecal) E. coli (AFEC), and atypical enteropathogenic E. coli (aEPEC).