Characterization of a plant pathogen-like type III secretion system in Burkholderia pseudomallei and identification of novel virulence factors in Burkholderia mallei

Abstract

Burkholderia pseudomallei and Burkholderia mallei are human pathogens that cause the diseases melioidosis and glanders, respectively. Type III secretion systems (T3SS) are specialized macromolecular machines that inject proteins from Gram negative bacterial cells directly into the cytosol of eukaryotes. B. pseudomallei possesses 3 distinct T3SSs. One is crucial for pathogenicity and similar to the T3SSs found in the animal pathogens Salmonella and Shigella. The other 2 T3SSs have no described role thus far and are similar to the T3SSs found in the plant pathogens Ralstonia solanacearum and Xanthomonas campestris. The focus here is one of the plant pathogen-like T3SSs (T3SS2). The studies presented in this dissertation explore the distribution, organization, regulation, and role of T3SS2 of B. pseudomallei. I found that orthologs to T3SS2 are found in 6 other Burkholderia species, including Burkholderia thailandensis, which we often used as a surrogate for B. pseudomallei. I found that T3SS2 in B. pseudomallei and B. thailandensis is positively regulated by an AraC type transcriptional regulator via a conserved DNA motif that is found in the promoter regions of T3SS2 genes. Since T3SS2 is similar to the T3SSs found in some plant pathogens we tested it’s involvment in phytopathogenicity and found that no disease-like symptoms were produced in three different plant species, making it highly unlikely that T3SS2 is involved in plant pathogenicity. To identify proteins that were potentially secreted by T3SS2 we performed shotgun proteomics on the culture supernatant proteins of B. pseudomallei overexpressing HrpBbp. Due to cell lysis, the majority of identified proteins were cytoplasmic which made identification of true T3SS2-secreted proteins nearly impossible. However, using a combination of transcriptomics and proteomics, 1 potential T3SS2-secreted protein was identified. While some of the factors critical for the virulence of B. mallei and B. pseudomallei have been described, more are yet to be discovered. Using in silico genome analysis, a list of potential virulence-related genes was generated for these bacteria. Some were tested for their virulence potential in a wax moth larvae model of infection developed in our lab. This resulted in the identification of 5 potential virulence factors.