UC San Diego

Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira, is a zoonotic infection that is a significant cause of worldwide morbidity and mortality for both humans and animals. Despite its global prevalence, the disease remains understudied, and the molecular mechanisms underlying its pathogenesis remain poor understood. Research has been hindered due to the slow growth of infectious isolates and their recalcitrance to genetic manipulation. This dissertation presents a pathogenomics-based approach for identifying novel virulence associated genes that couples deep sequencing analysis of experimentally attenuated highly pathogenic Leptospira interrogans serovar Lai with comparative analysis of the pan-genome of the entire Leptospira genus. In Chapter 2, this approach is used identify non- synonymous mutations in pathogen specific genes of L. interrogans that accumulated during the course of experimental attenuation. 11 genes were identified, and 10 of them are demonstrated to have profound in vivo transcriptional upregulation by wild-type virulent L. interrogans serovar Lai in an animal model of acute leptospirosis. Among these genes is a putative soluble adenylate cyclase, found in cell-free culture supernatant, that is shown to elevated cyclic-AMP levels in human monocytes in vitro. 2 genes were found to belong to a previously unstudied 15 member paralogous gene family shared among pathogenic Leptospira and two species of the alpha-proteobacterial genus Bartonella. How these distant species came to share this gene family remains an evolutionary mystery; however, Leptospira paralogs are shown to have unique tissue specific upregulation in vivo, suggesting a direct role in virulence. Chapter 3 demonstrates that a recombinant form of the adenylate cyclase protein recapitulates the host cyclic-AMP elevation observed in Leptospira culture supernatant. Chapter 4 presents analysis from a second experimentally attenuated L. interrogans serovar Lai strain. Improved bioinformatic and sequencing technology allow for a more in depth and comprehensive evaluation of mixed allele populations. This revealed that subpopulations of mutant non-synonymous alleles increased in frequency during the attenuation and were located in genes related to signal transduction mechanisms employed by the bacteria. The combined interpretation of the attenuation experiments and future directions for research are summarized in Chapter 5