Differential expression of virulence and stress fitness genes between Escherichia coli O157:H7 strains with clinical or bovine-biased genotypes

Abstract

Escherichia coli O157:H7 strains can be classified into different genotypes based on the presence of specific Shiga toxin-encoding bacteriophage insertion sites. Certain O157:H7 genotypes predominate among human clinical cases (clinical genotypes), while others are more frequently found in bovines (bovine-biased genotypes). To determine whether inherent differences in gene expression explain the variation in infectivity of these genotypes, we compared the expression patterns of clinical genotype 1 strains with those of bovine-biased genotype 5 strains using microarrays. Important O157:H7 virulence factors, including locus of enterocyte effacement genes, the enterohemolysin, and several pO157 genes, showed increased expression in the clinical versus bovine-biased genotypes. In contrast, genes essential for acid resistance (e.g., gadA, gadB, and gadC) and stress fitness were upregulated in bovine-biased genotype 5 strains. Increased expression of acid resistance genes was confirmed functionally using a model stomach assay, in which strains of bovine-biased genotype 5 had a 2-fold-higher survival rate than strains of clinical genotype 1. Overall, these results suggest that the increased prevalence of O157:H7 illness caused by clinical genotype 1 strains is due in part to the overexpression of key virulence genes. The bovine-biased genotype 5 strains, however, are more resistant to adverse environmental conditions, a characteristic that likely facilitates O157:H7 colonization of bovines.

FIG. 1.

Double loop design for the microarray experiment. B1 to B4 represent four different strains of the bovine-biased genotype 5, whereas C1 to C4 represent four different strains of the clinical genotype 1. Each arrow indicates a hybridization, with the arrowhead representing Cy3 and tail Cy5 dyes.

FIG. 2.

qRT-PCR validation of microarray data. The expression ratios between clinical and bovine-biased genotypes as calculated by microarray analyses and qRT-PCR are given. Results shown are average fold changes in expression, with SEM from four biological replicates (strains) per genotype.

FIG. 3.

Neighbor-joining phylogeny of SNP genotypes representing the eight strains examined in the study. Three of the four clinical genotype 1 strains belong to clade 8, whereas one clinical genotype 1 strain is part of clade 1. All four bovine-biased genotype 5 strains are members of clade 7. Clades were classified in a prior study, and control strains representing seven of the previously described clades (30) were used as a reference to categorize the clinical and bovine-biased genotypes.

FIG. 4.

Survival of clinical and bovine-biased genotypes in the model stomach system. The average survival rate (log decrease in CFU/ml/30 min) and the SEM from two independent experiments are plotted for each genotype.