Lyme disease, also known as Lyme borreliosis, a common vector-transmitted illness, caused throughout the globe by the pathogen Borrelia burgdorferi, which is transmitted by Ixodes scapularis ticks. In North America, I. scapularis transmits a wide array of human and animal pathogens including a group of pathogenic bacteria, known as B. burgdorferi sensu lato complex. Life cycle of B. burgdorferi primarily involves an intricate tick-mammal infection cycle. It transits between different hosts, an arthropod vector and a variety of vertebrate hosts. Maintenance of B. burgdorferi in the enzootic cycle requires successful persistence in the arthropod and reservoir hosts, as well as efficient transmission between tick and mammalian host. Therefore, in order to survive transitions between diverse host-vector environments, B. burgdorferi must not only be able to detect changes in its environment, but also generate suitable response to these changes. As a result, gene-products playing roles in adaptation to stress, including temperature, oxidative stress, pH etc. must be critical for the maintenance of life cycle of the pathogen. One such gene product, which is very important for the bacterial adaptation of stress, is the High temperature requirement protease A (HtrA). HtrAs in different bacteria primarily function in protein homeostasis and quality control, acting as protease and chaperone for stabilizing specific proteins and modulate signaling pathways. While other bacteria like Escherichia coli or other spirochetes like Leptospira possess multiple homologs of HtrA, B. burgdorferi genome harbors a single HtrA gene; which was first described from our laboratory and termed as BbHtrA. The primary goal for this dissertation is to characterize the function of BbHtrA and to study the physiological relevance of this protease during pathogenesis of Lyme disease. Utilizing BbHtrA mutant we studied the biological relevance of this protease on B. burgdorferi survival at higher temperatures and the effects of its deletion on different virulence determinants. Key areas of this research involves a better understanding of intriguing biology and infection of B. burgdorferi, including identification of novel virulence factors which will help and contribute to the development of new strategies that interfere with pathogen persistence and transmission.