Thomassin, Jenny-Lee2024-06-1120242024-112024-06-11November 2https://hdl.handle.net/10388/15756Citrobacter rodentium, a Gram-negative bacterium, is a murine pathogen that is used as a surrogate model for the human pathogen enterohemorrhagic Escherichia coli (EHEC). Both of these pathogens share many virulence factors and cause similar infections. Among the shared virulence factors are specialized secretion systems that secrete proteins involved in pathogenesis. One secretion system that is found in EHEC and might also be used by C. rodentium is the type II secretion system (T2SS). The T2SS in C. rodentium had not been previously characterized, therefore the overall aim of this research was to characterize T2SS from C. rodentium and to identify the in vitro conditions that promote T2SS-activation. First, a bioinformatic analysis of the T2SS was performed and homologs of all genes that have been shown to be necessary in extensively studied E. coli model systems were found in the genome of C. rodentium. In addition, in silico analysis predicted the presence of two putative promoters, one at the beginning of each operon encoding T2SS genes, and two terminators, one at the end of each operon. Next, promoter activity was monitored by transcriptional lacZ fusion assays. The T2SS-promoter activity was also assessed by transcriptional fluorescence fusion assays. These data indicate low-level constitutive activity of the T2SS promoter under nearly all growth conditions tested. In addition, secretion assays were performed under various growth conditions to identify T2S proteins. It was discovered that the growth of C. rodentium in a minimal medium before subculturing in TSB media led to the presence of some protein species in the supernatant of wild-type C. rodentium that were absent from that of a T2SS deletion strain. Identification of the novel protein species by mass spectrometry revealed the presence of proteases and two lysogenic bacteriophages. Taken together, these data provide the first evidence that the general secretory pathway (gsp) T2SS in C. rodentium is active and functional for protein secretion. Future work will focus on validating whether the identified esterase is a T2SS substrate and whether the lysogenic bacteriophage hijacks the C. rodentium T2SS secretin for release as was previously observed for the CTXφ lysogenic bacteriophage of V. cholerae.application/pdfenBacteriaProtein secretion ssytemType 2 secretion systemInfectionThesis2024-06-11