Studies on pathogenic bacteria using genetic and genomic approaches and a novel small animal model

Abstract

Infections with pathogenic bacteria result in disease in both humans and animals. Bacteria rely on virulence factors to cause disease in their hosts. Deepening our understanding of host-pathogen interactions requires identification and characterization of bacterial virulence factors as well as analysis of their roles in infection in vivo. Genetic and genomic approaches serve as powerful, high-throughput approaches to define these factors, while dissection of their in vivo roles requires a relevant small animal model of infection. In this thesis, I describe our work on two bacterial pathogens of global importance, Streptococcus equi subspecies zooepidemicus (SEZ), a scourge of swine, and Shigella, the current leading bacterial cause of diarrheal deaths worldwide. In SEZ, a combined genetics and genomics approach led to the discovery of a novel virulence regulator, SezV, that activates expression of a neighboring gene that is a key virulence factor and immunogen, the M-like protein, SzM. Absence of SezV led to a severe attenuation of virulence in a mouse infection model. Large-scale analyses of publicly available genomes revealed that the sezV szM locus was present in all strains of SEZ and the closely related bacteria, S. equi subspecies equi, and some S. pyogenes strains, defining a distinct class of virulent streptococci. Furthermore, we found evidence that a specific monoclonal antibody to a common microbial surface polysaccharide, poly-N-acetylglucosamine (PNAG) recognized the SzM protein, suggestive of protein glycosylation. In our work on Shigella, we developed a small animal model of infection involving oral inoculation of infant rabbits. Infected animals developed disease with diarrhea and intestinal pathology reminiscent of aspects of human disease. The bacteria robustly colonized the intestine, invaded colonic epithelial cells, and foci of invasion, which represent sites where several neighboring epithelial cells contained intracellular bacteria, were observed in the colon. In situ mRNA labeling demonstrated that expression of a key chemokine, IL-8, was elevated in uninfected cells near infected cells. Finally, we found that two key Shigella virulence factors, IcsA, a protein required for cell-to-cell spreading, and the type III secretion system (T3SS), were required for the development of diarrhea and intestinal pathology.