Clonal isolation of Borrelia miyamotoi identifies multiple infection phenotypes and potential novel virulence determinants

ABSTRACT Borrelia miyamotoi is an emerging tick-borne pathogen in the Northern Hemisphere that causes a relapsing fever-like illness known as Borrelia miyamotoi disease. Despite increased recognition of B. miyamotoi as a human pathogen, the mechanisms underlying infection and pathology remain poorly understood. During routine cultivation of B. miyamotoi LB-2001, our lab observed that bacterial cultures extensively passaged in vitro (over 70 passages) lost the ability to establish infection in severe combined immunodeficiency (SCID) mice. Clonal populations were isolated from both high- and low-passage cultures, and three distinct phenotypes were emerged—infections, attenuated, and non-infectious—each showing an inverse correlation between culture growth and infectivity in SCID mice. Infection outcomes in mice revealed differences in spirochetemia, hepatosplenomegaly, and anemia among the clones. Genomic DNA sequencing identified potential virulence determinants associated with these infectious phenotypes. These findings lay the groundwork for uncovering molecular mechanisms of B. miyamotoi pathogenesis and identifying targets to mitigate the impact of this emerging pathogen. IMPORTANCE As global expansion of tick habitats increases, tick-borne diseases are becoming more prevalent and widespread. The emerging tick-borne pathogen Borrelia miyamotoi warrants increased scrutiny. This work demonstrates loss of infectivity in immunocompromised mice by B. miyamotoi subjected to continual in vitro cultivation, as well as the isolation of clonal populations that demonstrate differences in infectivity with three distinct phenotypes: infectious, attenuated, and non-infectious. Characterizing how clonal isolates have differences in infectivity can lead to the identification of novel factors required for virulence.