The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology

Abstract

<u>R</u>elapsing <u>f</u>ever (RF), caused by spirochetes of the genus <i>Borrelia</i>, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF <i>Borrelia</i> spirochetes are unknown. <u>C</u>yclic <u>di</u>-<u>AMP</u> (c-di-AMP), synthesized by <u>d</u>i<u>a</u>denylate <u>c</u>yclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although <i>Borrelia</i> is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium <i>Borrelia turicatae</i>, a <i>cdaA</i> mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on <i>cdaA</i> mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of <i>cdaA</i> confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of <i>cdaA</i> did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium <i>Borrelia burgdorferi</i> Finally, the <i>cdaA</i> mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to <i>cdaA</i> inactivation. Overall, these results indicate CdaA is critical for <i>B. turicatae</i> pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.