| Burkholderia pseudomallei is the etiological agent of melioidosis, a serious global emerging infectious disease. Within the context of host-cell infection, this category B potential bioterrorism agent infects various cell types by going through different stages: i) entry into endocytic vesicles, ii) escape from these vesicles into the cytoplasm, iii) intracytoplasmic replication, iv) polymerization of cytoplasmic host-cell actin, and v) catapulting themselves directly into neighboring cells through membrane protrusions to spread from cell to cell. We hypothesized that B. pseudomallei, as it encounters these different in tracellular environments and perform sequential steps in the infection process, undergoes differential gene expression at each of these stages.;To test this central hypothesis, I have initially developed various genetic tools for routine genome manipulations in B. pseudomallei, including reporter-gene fusions, gene knocking-out, and complementations. Consequently, a single-bacterium isolation and total transcript amplification method was demonstrated and validation of this single cell method was done via reporter-gene fusion studies using the newly developed genetic tools. This single cell total transcript amplification method was then utilized to obtain genome-wide gene-expression profiles in individual B. pseudomallei cells isolated from different macrophage compartments. Differential gene expression was observed in a spatially dependent manner. As a proof of concept, two in vivo induced genes (one in macrophage vacuole and the other in cytoplasm during actin polymerization) were then mutated to confirm their involvement in these corresponding intracellular infection processes. With these data, I have identified differentially expressed genes including many potential virulence genes, and also provided a better understanding of bacterial response to the different host environment. Furthermore, these data allowed assignment of many putative and hypothetical proteins to important pathophysiological events during B. pseudomallei host-cell infection. |
