| dc.description.abstract | Chlamydia are unique obligate intracellular organisms responsible for disease in a wide variety ofhosts. A defining feature of these bacteria is their biphasic developmental cycle, through which Chlamydia interact with the host cytoplasm using surface proteins and secreted effectors. As a major public health pathogen, Chlamydia trachomatis is the causative agent behind nearly two million sexually-transmitted infections in the United States every year. Symptoms of chlamydia can be permanent and life-threatening, including infertility, ectopic pregnancy and pelvic inflammatory disease, although for many, infections are asymptomatic. There remain many mysteries about how Chlamydia are able to cause disease in some patients while remaining unnoticeable in others. Prominently among these mysteries is how Chlamydia undergo ascension, or disseminate to the fallopian tubes to cause these complicated sequelae. Importantly, mouse models for C. trachomatis are limited, as C. trachomatis is an obligate human pathogen, unable to mimic human pathogenesis in the mouse. Closely related C. muridarum, an obligate mouse pathogen, shares 99% of genetic content with C. trachomatis. With this dissertation, I seek to answer how species with such genomic conservation can be host-specific. Host-specificity is common amongst bacteria and is often mediated by factors such as surface receptors, immune evasion effectors and nutrient acquisition components. In Chlamydia, very few of these factors have been identified. Therefore, I used comparative genomics and chimeric recombinants to investigate the host-specificity of Chlamydia. Through the complete genome sequencing and annotation of Chlamydia suis, a close relative of both C. trachomatis and C. muridarum, I demonstrated that C. suis contains a subset of unique proteins, as well as shared putative host-interacting proteins with those found in C. muridarum and/or C. trachomatis. C. muridarum and C. trachomatis genomes were crossed to generate a library of chimeras where C. muridarum genes were integrated into the C. trachomatis genome using homologous recombination. With these chimeras, I investigated the role of C. muridarum genes on pathogenesis phenotypes. First, I focused on the plasticity zone, a region of diversity in Chlamydia, thought to be important for mouse infections and host cell toxicity in vitro. Replacement of C. trachomatis plasticity zone genes with C. muridarum genes showed that the plasticity zone genes are partially involved in regulating the developmental cycle and inclusion morphology, as some plasticity zone chimeras had reduced progeny 36 hours post-infection and significantly smaller inclusion sizes than wildtype C. trachomatis. In the mouse model, C. muridarum plasticity zone genes did not enable the recombinants to ascend to upper reproductive tissues, nor were they more successful in avoiding clearance or establishing increased bacterial burdens in the lower reproductive tract than C. trachomatis. Three genes within the C. muridarum plasticity zone, referred to as large putative cytotoxins, which have long been considered responsible for an in vitro host cell toxicity phenotype of C. muridarum, were shown to be insufficient for this phenotype and additional genes may be involved. Additionally, an inclusion membrane protein from C. muridarum was unable to perform the function of the C. trachomatis ortholog, suggesting that this protein interacts with another chlamydial protein to stabilize the inclusion. Second, chimeric recombinants across the genome were examined in a growth curve screen where two unique growth phenotypes were discovered; a poor growing mutant and a mutant which produces infectious progeny faster than C. trachomatis. Regions of recombination for these chimeras suggest candidate genes involved in these phenotypes. Mouse models for these recombinants reveal that C. muridarum pathogenesis and ascension in the mouse is likely multifactorial as no recombinant was capable of ascension. Taken together, I have used comparative genomics and recombinant libraries to increase our knowledge of the plasticity zone, demonstrate the use of recombination in Chlamydia as a screening tool for host-specific phenotypes and suggest that C. muridarum and C. trachomatis orthologs are largely interchangeable, but that small coding changes can lead to biologically relevant phenotypes. | |
