|Institution:||Washington State University|
|Keywords:||Microbiology; Molecular biology; ExsE; parahaemolyticus; T3SS; Transcriptome; Vibrio; Virulence|
|Full text PDF:||http://hdl.handle.net/2376/5090|
Vibrio parahaemolyticus is a bacterial pathogen capable of causing gastroenteritis, wound infections and septicemia. Its virulence factors include two type III secretion systems (T3SS1 and T3SS2) that cause host-cell cytotoxicity and enterotoxicity, respectively. This dissertation presents analysis of the genome of V. parahaemolyticus to identify T3SS associated genes, construction of comparative transcriptomic profiles of T3SS1 activity, and targeted analysis of T3SS1 regulatory gene exsE. To identify secreted proteins, chimeric fusions were constructed between putative effector protein leader sequences and a leaderless phospholipase, yplA. These constructs were subsequently expressed using the heterologous host Yersinia enterocolitica. Screens included genes from T3SS1 and T3SS2 associated regions and the entire V. parahaemolyticus genome; several T3SS1 and T3SS2 secreted proteins were identified, as well as seven additional genes located elsewhere in the genome. To identify genes involved in cytotoxicity, a mini-Tn5 transposon insertional knockout library was generated and screened for loss of T3SS1-dependent cytotoxicity towards HeLa cells. Thirty-six insertional mutants were identified; 17 were associated with T3SS1 and 17 unique genes were identified that may contribute to T3SS1-dependent cytotoxicity. ExsE is an important regulatory protein for T3SS1; deletion of exsE did not affect T3SS1 transcription or synthesis, but it did result in attenuated cytotoxicity towards HeLa cells characterized by reduced autophagy and decreased adherence to host cells. However, exsE deletion had no detectable effect on T3SS1-dependent mortality using an intrapulmonary infection of mice, suggesting other regulatory pathways may be involved during in vivo infection. Comparative transcriptional profiles of V. parahaemolyticus were assembled from T3SS1 inducing (DMEM induction, exsA overexpression, HeLa cell infection) and non-inducing (LB-S, exsD overexpression) conditions, which revealed differences in transcription of iron acquisition genes between DMEM and exsA induction. These types of genes also predominate early during HeLa infection but are overshadowed by nitrate and inorganic transport genes by mid- to late-infection. Expression patterns of T3SS1 associated genes were also analyzed over the course of HeLa cell infection. Thirteen unique upregulated genes were identified in common between DMEM and exsA induction, and 33 unique genes were identified that showed upregulation over the course of HeLa infection.