Grant: Discovery Grants Program - Individual

Adaptation of bacteria to new host environments is critical for their survival, growth, and behaviour. For many bacteria and, in particular, for Shigella, the evolution of unique adaptive capabilities remains poorly understood. Bacterial evolution proceeds through de novo mutation, horizontal gene acquisition and gene loss. When bacteria acquire new genetic material, significant costs may be imposed if the new genes are inappropriately controlled. Genes that interfere with an acquired virulence program are termed "antivirulence genes" and they can be selected against during incipient host adaptation. Identifying antivirulence genes has, to date, been accomplished in a haphazard manner and this proposal seeks to develop a rational, phylogenetically supported method for identifying and verifying these genes in Shigella. The human-adapated pathogen, Shigella, provides a unique opportunity to understand antivirulence gene loss because these bacteria have evolved independently and on multiple occasions. Following the acquisition of the invasion plasmid, each lineage converged on the Shigella-like phenotype through independent parallel evolution. As each new acquisition provides an opportunity for a new Shigella-like strain to evolve, we hypothesize that genes that have been independently lost from modern lineages of Shigella represent previously unrecognized antivirulence genes. We describe a research program that rationally identifies candidate antivirulence genes and tests the role of each in Shigella behaviour. Using publicly available genome sequences, we examined all of the  regulatory systems of E. coli in Shigella. We mapped each gene loss event against the evolutionary pattern of Shigella emergence, and identified independent regulatory gene loss events. We found that one or more components of the pmrDAB, citAB, kdpDE, ycaN or ybhD system have been lost. Importantly, known Shigella virulence regulators (phoPQ, ompR-envZ) are not pseudogenized in any lineage. One of the identified systems, PmrDAB, regulates resistance to antimicrobial peptides produced during host infection and is important for pathogenesis in many bacterial species. In 8/10 lineages of Shigella, one or more of the genes are nonfunctional. We show that restoring function of PmrD to S. flexneri restored antimicrobial peptide resistance but interfered with the normal regulation of a key invasion plasmid  type III secretion system. We will test how restoration of each system to Shigella alters bacterial physiology, growth and interaction with host cells. This rational, phylogenetically informed approach to identifying and characterizing antivirulence gene loss will be useful for understanding other bacterial species that have followed a similar evolutionary trajectory. In parallel, we will also examine whether deleting the identified genes from a newly emergent enteroinvasive E. coli strain enhances host-pathogen interaction in a manner consistent with this hypothesis.