Borrelia burgdorferi bb0164 encodes a Na+/Ca2+ antiporter homolog with a novel role in Mn2+ homeostasis and infectivity

Borrelia burgdorferi, the Lyme disease causative agent, relies on trace metals for motility, growth, and virulence in the absence of metal transport homologues encoded in the genome. Previous studies characterized borrelial metal transporter (bmtA) as a manganese (Mn) transporter and speculated that it is the sole Mn transporter. An oxidative stress transposon library screen identified bb0164, annotated as a calcium/sodium antiporter, as having a putative metal binding domain. The transposon mutant lost the ability to internalize Mn suggesting B. burgdorferi is using a non-canonical protein for metal transport. In this study, a bb0164 deletion and complement were generated in B. burgdorferi 5A4-NP1 to evaluate for trace metal transport, virulence regulation, resistance to oxidative stress, and infectivity. Our data demonstrated that the loss of bb0164 resulted in a significant reduction in internalized Mn, increased sensitivity to oxidative stress, dysregulation of the BosR-RpoS virulence pathway, and a loss of infectivity in mice. The loss of bb0164 resulted in elevated rpoS, ospC, and dbpA expression and production, while bosR was not altered transcriptionally or post-transcriptionally. B. burgdorferi grown in chelated complete BSK-II media showed a similar sensitivity to oxidative stress and virulence dysregulation as the bb0164 mutant. These phenotypes were rescued by exogenous Mn and Zn without influencing the expression levels of bb0164 or bmtA. AlphaFold models of BB0164 were structurally divergent from the canonical bacterial Mn transporter, Bacillus subtilis MntH and B. burgdorferi BmtA, but shared high similarity with a calcium/cation antiporter superfamily member. Together, this study characterized BB0164 as a second non-canonical Mn transporter in B. burgdorferi that is essential for mammalian pathogenesis and likely supports metal homeostasis along with bmtA. More broadly, B. burgdorferi uses unique and uncharacterized mechanisms for metal homeostasis that supports physiology and pathogenesis of the spirochete during mammalian infection. Author SummaryLyme disease, caused by Borrelia burgdorferi, is the most common vector-borne illness in the United States and can result in a chronic inflammatory disease. B. burgdorferi acquires most of the necessary nutrients, including trace metals, from the host due to its limited metabolic capacity. B. burgdorferi has evolved a manganese-centric metabolism in place of the iron primarily used by other bacteria. Little is understood about manganese homeostasis in B. burgdorferi with a single transporter, BmtA, characterized to date. Here, we describe a second manganese transporter, encoded by bb0164, that is essential for infection, protects against oxidative stress, and alters genetic regulation. We found that BB0164, an annotated ion antiporter, does not structurally align with conserved manganese transporters from other bacteria. Interestingly, our work suggests bb0164 and bmtA are not subject to transcriptional regulation dependent on temperature or metal availability, differing from other bacterial manganese transporters. These findings indicate B. burgdorferi is uniquely using an antiporter protein for metal transport to support metal homeostasis, which further demonstrates the importance of manganese in borrelial pathogenesis.