Repeated Loss of Functional PMRDAB Signaling Systems in Shigella Flexneri Due to Altered Regulation of the MXI-SPA Type 3 Secretion System

Shigella spp has evolved on multiple occasions following the acquisition of a large virulence plasmid encoding a dedicated type III secretion system. Following the acquisition of these genes and others, the bacteria have undergone the repeated and independent loss of other core genes that interfere with this newly acquired virulence program. We have observed that genes encoding components of the PmrD-PmrAB signaling systems appear to have been repeatedly lost in Shigella spp. In this work, we investigate the consequences of the loss of functional PmrD from S. flexneri strains. This PmrD protein is a connector protein that links PhoPQ and PmrAB signaling systems together, and this connection is important for the appropriate regulation of LPS modifications, antimicrobial peptide resistance, and virulence in Salmonella enterica and E. coli. In Shigella spp, the T3SS is regulated by a number of different regulators, especially the master regulator, VirF. The promoter for the virF gene houses a putative PmrA binding site. Here, I demonstrate that restoring functional pmrD in trans in S. flexneri M90T shows repression of T3SS under low Mg2+ conditions. Furthermore, I show that independent iron-mediated signaling through the PmrAB system shows ~50% repression of T3SS. I also show that active PmrA significantly decreases T3SS expression. Finally, by making chromosomally integrated functional pmrD alleles, we show that virulence of S. flexneri is dramatically lost in a Galleria mellonella model of infection. These results are consistent with a model where the PmrDAB system interferes with normal regulation of the T3SS and therefore pmrD is an antivirulence gene in S. flexneri.