Gain-of-function study reveals the pleiotropic roles of serine protease HtrA in Borrelia burgdorferi

High-temperature requirement protease A (HtrA) is a family of serine proteases degrading misfolded and damaged proteins that are toxic to bacteria. The Lyme disease agent Borrelia burgdorferi encodes a single HtrA (BbHtrA). Previous studies have shown that BbHtrA is a key virulence determinant of B. burgdorferi as a deletion mutant of htrA ({Delta}htrA) fails to establish infection in mice. However, previous complementation could only restore protein expression but not infectivity in mice. In this report, we first identify the native promoter of BbHtrA which allows us to construct a fully complemented{Delta} htrA strain. Follow up promoter activity analysis reveals that BbHtrA is likely dually regulated by the house keeping sigma factor RpoD and the alternative sigma factor RpoS. The{Delta} htrA mutant exhibits growth defect upon entering the mid-log to stationary phase especially at high temperatures. Microscopic analysis further demonstrates that the absence of htrA induces extensive cell death. Additionally, the{Delta} htrA mutant has defects in cell locomotion as the expression of several key chemotaxis proteins are significantly downregulated. Cryo-electron tomography imaging of htrA mutant further reveals that deletion of htrA disrupts flagellar homeostasis. The failure of{Delta} htrA to establish an infection in mice is likely due to repressed expression of BosR and RpoS at the transcriptional level which ultimately causes dysregulation of the RpoS-induced virulence factors. Collectively, we conclude that the expression of htrA is finely tuned which is critical for its pleiotropic roles in the regulation of motility, stress response, and virulence gene expression in B. burgdorferi. IMPORTANCELyme borreliosis is the most commonly reported vector-borne illnesses in the United States, which is caused by Borrelia burgdorferi. As the enzootic pathogen alternates between the tick vector and mammalian hosts, adaptation to drastically different growth milieu is imperative to its survival. Hence, robust alteration of gene expression and proper quality control on protein synthesis and turnover are pivotal for its fitness. The family of HtrA serine proteases is mainly responsible for the maintenance of protein homeostasis particularly under stressful conditions. The significance of this report is to decode how BbHtrA contributes to the fitness of B. burgdorferi. BbHtrA is essential for mammalian host infection but little is known about its regulatory mechanism as well as its contribution to the virulence of B. burgdorferi. By deciphering the regulatory elements involved in the expression of BbHtrA, we are one step closer to comprehending its significance in the pathophysiology of B. burgdorferi.