Theregulatory elements in: a novel small RNA impacts gene expression and mammalian infection.

Abstract

UNLABELLED: The segmented genome of, the tick-borne agent of Lyme disease, encodes numerous chromosomal and plasmid-borne proteins and small RNAs (sRNAs) of unknown function that are critical for infectivity. Two recent examples are the linear plasmid (lp)17-encoded protein BBD18 and sRNA(also termed as), which promote spirochete survival in ticks and mammals, respectively. Using targeted mutagenesis of thelocus, complementation, and phenotypic analysis of isogenic mutants, we herein confirm and extend the regulatory roles of BBD18 and(). A mutant lacking BBD18 and() persisted in ticks, yet failed to infect immunocompetent or immunodeficient mice. Althoughcomplementation selectively restoredexpression, it did not rescue murine infectivity, supporting an essential role for() during mammalian infection. Transcriptomic and proteomic analyses revealed widespread alterations in expression profiles that were only partially rescued bycomplementation, suggesting distinct regulatory functions for BBD18 and(). Because an additional sRNA () lies immediately downstream relative to, we generated an isogenicinactivation mutant, which was likewise largely noninfectious in mice and exhibited dysregulation of multiple gene products, including the induction of severalgenes, such as, and the downregulation of multiple proteins, such as OspC, BamA, and DbpA. Together, these data indicate that thelocus is surrounded by two essential sRNA elements,and(), all three of which independently regulate genes, including ones impacting mammalian infectivity. Further characterization of such atypical regulatory elements inmay inform new control strategies. IMPORTANCE: , the tick-borne agent of Lyme disease, is the causative agent of one of the most prevalent vector-borne infections in many regions worldwide. Despite extensive study, the biological functions of many of its protein and small RNA (sRNA) products remain poorly defined. Here, we confirm and extend the regulatory roles of the linear plasmid ()17-encoded protein BBD18 and the sRNA() in spirochete infectivity. Importantly, we identify a previously unrecognized regulatory function for an adjacent sRNA,, underscoring lp17 as a key regulatory region in. Together, our findings highlight thelocus and its surrounding sRNA elements as an independent, multilayered regulatory module that controls gene products, including those required for mammalian infection. Defining how these three regulators shape gene expression and virulence will reveal new mechanisms underlying Lyme disease pathogenesis and may inform the development of new strategies to prevent this widespread illness.