Signalling System Heterogeneity in E. coli and Shigella

Candidate: Raymond Huynh

Supervisor: Dr. Joseph McPhee

Abstract:

Many organisms produce host defense peptides (HDPs). These molecules protect against invading or competing microbes by either killing or slowing down the growth of the opposing microorganism as well as having immune signaling effects. In response to these antibacterial effects, many bacteria have adapted to these changing environmental conditions and stressors by employing regulatory systems to resist antimicrobial HDP activity. In Enterobacterales, signaling systems such as the PhoPQ and PmrAB two component systems (TCS) are the most important drivers of HDP resistance. These systems regulate genes that are involved in bacterial HDP resistance, principally by modifying the surface properties of the bacterium such that their interaction with HDPs is reduced and bacteria are protected from it.

The connector protein, PmrD links the PhoPQ and PmrAB TCS by preventing dephosphorylation of the response regulator, PmrA through PhoPQ mediated signaling and is critical for PhoPQ-regulated HDP resistance in both Escherichia coli and in Salmonella enterica. In E. coli (and closely related Shigella spp.) the pmrD gene contains three putative start codons near a Shine-Dalgarno sequence and E. coli full-length in-frame pmrD is produced from the second putative translational start site. In S. flexneri, the pmrD gene contains a 1 bp insertion in the 5' end of the gene, bringing the full-length ORF in frame with the first putative start site, Earlier work has demonstrated that only one of the three putative translational start sites found within Escherichia coli results in expression and production of functional PmrD protein. Here, I demonstrate that the start site spacing found in Shigella flexneri does not produce PmrD and that this organism does not undergo low Mg2+ regulated polymyxin B resistance. Complementation with the E. coli pmrD orthologue restores canonical signaling and regulation of resistance. These findings suggest that unlike E. coli, S. flexneri resist HDP independent of the PmrD-axis, indicating other regulatory networks may be involved in high level induction of HDP resistance observed within S. flexneri.

Although non-functional in S. flexneri, I further show that PmrD in S. sonnei is like that from E. coli and is required for high level PhoPQ-regulated polymyxin B resistance in this organism, suggesting that these species differ in this signaling network. My results establish that Shigella spp. are heterogenous in their regulation of polymyxin B resistance, suggesting that the loss of functional pmrD in S. flexneri may represent a species-specific pathoadaptation.