Characterization of proximity-dependent inhibition: The discovery of the novel microcin, MccPDI.

摘要

The strain E. coli-25 was originally isolated from cattle at the WSU dairy and was later discovered to exhibit an inhibitory phenotype in vitro. This phenotype was designated proximity-dependent inhibition (PDI) and allowed E. coli-25 to restrict the growth of a broad range of E. coli strains, including enterohemorrhagic E. coli O157:H7 and E. coli O26. Next-generation sequencing identified candidate plasmid-borne genes that encoded a putative microcin, later designated as MccPDI. Five genes (mcpM, mcpI, mcpA, mcpB, and mcpD) were present in the MccPDI gene cluster and were necessary for microcin production, self-immunity, and export. In addition, the chromosomally located tolC was also required for inhibition.;The genes necessary for susceptible E. coli to succumb to inhibition by MccPD were determined by screening a single-gene knockout library of E. coli strain BW25113 for sensitivity to MccPDI. Six genes were identified that are required for inhibition by MccPDI ( atpA, atpB, dsbA, dsbB, ompF, and ompR). OmpF is hypothesized to be the receptor that allows MccPDI to bind to the surface of susceptible E. coli. The other proteins are likely required for the expression and function of OmpF or are necessary to translocate MccPDI into the cell.;Because E. coli-25 was able to inhibit the growth of human pathogenic E. coli in vitro, MccPDI-producing E. coli and non-producing E. coli strains were tested for their ability to limit the colonization of susceptible E. coli in vivo. Neonatal calves were co-inoculated with a mixture of either MccPDI-producing or non-producing E. coli-25 in conjunction with either susceptible E. coli-186 or E. coli O157:H7. During the in vivo trials, MccPDI-producing E. coli strain composed a higher percentage of the total population of lactose fermenting bacteria compared to the non-producing strain. The MccPDI-producing E. coli strain out-competed susceptible E. coli-186, but was unable to out-compete co-inoculated E. coli O157:H7 in calves. These results indicate that MccPDI is functional in vivo and likely contributes to the fitness of E. coli-25 within a calf, although pre-colonization may be needed to inhibit subsequent colonization by E. coli O157:H7.