A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA(1) and CheA(2). Our previous study showed that CheA(2) is a genuine CHK that is required for chemotaxis; however, the role of CheA(1) remains mysterious. This report first compares the structural features that differentiate CheA(1) and CheA(2) and then provides evidence to show that CheA(1) is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA(1) has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA(1) is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA(1) (cheA(1)(mut)) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA(1) is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA(1) affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA(1)(mut) fails to elicit mouse tnf-alpha, il-10, il-1 beta, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA(1) in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.