Non-pathogenic Escherichia coli Enhance Stx2a Production of E. coli O157:H7 Through Both bamA-Dependent and Independent Mechanisms

Abstract

Intestinal colonization by the foodborne pathogen Escherichia coli O157:H7 leads to serious disease symptoms, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Synthesis of one or more Shiga toxins (Stx) is essential for HUS and HC development. The genes encoding Stx, including Stx2a, are found within a lambdoid prophage integrated in the E. coli O157:H7 chromosome. Enhanced Stx2a expression was reported when specific non-pathogenic E. coli strains were co-cultured with E. coli O157:H7, and it was hypothesized that this phenotype required the non-pathogenic E. coli to be sensitive to stx-converting phage infection. We tested this hypothesis by generating phage resistant non-pathogenic E. coli strains where bamA (an essential gene and Stx phage receptor) was replaced with an ortholog from other species. Such heterologous gene replacement abolished the ability of the laboratory strain E. coli C600 to enhance toxin production when co-cultured with E. coli O157:H7 strain PA2, which belongs to the hypervirulent clade 8. The extracellular loops of BamA (loop 4, 6, 7) were further shown to be important for infection by stx2a-converting phages. However, similar gene replacement in another commensal E. coli, designated 1.1954, revealed a bamA-independent mechanism for toxin amplification. Toxin enhancement by 1.1954 was not the result of phage infection through an alternative receptor (LamB or FadL), lysogen formation by stx2a-converting phages, or the production of a secreted molecule. Collectively, these data suggest that non-pathogenic E. coli can enhance toxin production through at least two mechanisms.

A putative microcin amplifies Shiga toxin 2a production ofEscherichia coliO157:H7

“…In mice given a co-culture of O157:H7 and phage-resistant E. coli , minimal toxin was recovered in the feces, but with E. coli that were phage-susceptible, higher levels of toxin were found (46). However, it is clear that phage infection of susceptible bacteria is not the only mechanism by which the gut microflora affects Stx2 levels during infection (19, 20, 24, 26).…”

“…E. coli 0.1229 produces at least two molecules capable of increasing Stx2a. The first is MccB17, a DNA gyrase inhibitor, shown to activate Stx2a production in an earlier study (24). This current study identified a second molecule localized to a 12.8 kb plasmid, and all genes necessary for production are found within a 5.2 kb region.…”

“…Conversely, non-pathogenic E. coli that are susceptible to infection by the stx2a -converting phage were reported to increase Stx2a levels (22, 23). This mechanism is O157:H7 strain-dependent (23), and requires expression of the E. coli BamA, which is the phage receptor (24, 25).…”

“…Production of Stx2a by O157:H7 can also increase in response to molecules secreted by other members of the gut microbiota (24, 26), such as bacteriocins and microcins. Bacteriocins are proteinaceous toxins produced by bacteria that inhibit the growth of closely related bacteria.…”

“…In support of this, the addition of extracted DNase colicins to various E. coli O157:H7 strains increased Stx2a production, but not Stx1 (26). Additionally, microcin B17 (MccB17), a DNA gyrase inhibitor, was shown to amplify Stx2a production (24).…”