Loss of functional pmrA as a pathoadaptation in the Shigella spp.

Candidate: Arooj Qamar
Supervisor: Dr. Joseph McPhee

ABSTRACT

Shigella spp. are pathogens responsible for the global burden of diarrhoeal disease, known to significantly affect children younger than 5 years and elderly populations. The genus is comprised of four species: Shigella sonnei, Shigella boydii, Shigella flexneri and Shigella dysenteriae, each phylogenetically different but phenotypically and pathogenically similar. Conserved in many pathogenic Gramnegative bacteria, is the connector protein, PmrD which links the PhoPQ and PmrAB two component systems which is critical for regulated host defense peptide resistance, and to achieve optimal virulence (via the T3SS) during different stages of pathogenesis. Previous work in the McPhee lab identified a mutation in pmrD which resulted in the loss of PmrD protein in S. flexneri M90T. Here, I study the hypothesis of whether there is loss of PmrD-PmrAB signalling cascade in other strains of Shigella besides S. flexneri. To do so, I first investigated their genomes: I look at the evolutionary relationship between Shigella spp and their close relative Escherichia coli and, inspect for genomic variance e.g., nucleotide insertions, deletions, or single nucleotide polymorphisms (SNPs), which may lead to pathoadaptation specifically in the PmrDPmrA- PmrB signalling cascade conserved in many pathogenic Gram-negative bacteria. Using pangenome analysis we construct a core-gene phylogeny and find our results in agreement with previous findings that Shigella spp has multiple origins of evolution. In our work we also show their genomes spread over five prominent clusters, interspersed within Escherichia genomes. We further look for genomic variance in pmrD and pmrA genes in each cluster. We identify 11 distinct genomic variations of pmrD and pmrA genes in total; 4 of pmrD and 7 of pmrA. From here we conducted in vitro microbiology experiments of Shigella sp. containing specific gene variants of pmrA and of pmrD and find that a truncated pmrA present in a subset of S. dysenteriae strains did not maintain PmrAB-regulated polymyxin B resistance. We further go on to work with a dominant pmrD variant present in all S. flexneri strains and the truncated pmrA present in some S. dysenteriae strains, to show that the PmrD-PmrA-PmrB signalling cascade does not modulate O antigen length in our S. flexneri or S. dysenteriae gene variants. We conclude that although mutations in PmrD-PmrA-PmrB doesn’t alter the O antigen length, they might be affecting other aspects of host-pathogen interaction.