The Intergenic Small Non-Coding RNA ittA is Required for Optimal Infectivity and Tissue Tropism in Borrelia burgdorferi

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed <jats:italic>trans</jats:italic>-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the <jats:italic>Borrelia burgdorferi</jats:italic> Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic <jats:italic>trans</jats:italic>-acting sRNA, which we designate here as <jats:italic>ittA</jats:italic> for <jats:italic>i</jats:italic>nfectivity-associated and <jats:italic>t</jats:italic>issue-<jats:italic>t</jats:italic>ropic sRNA locus <jats:italic>A</jats:italic>. The genetic inactivation of <jats:italic>ittA</jats:italic> resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the <jats:italic>ittA</jats:italic> mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for <jats:italic>ittA</jats:italic> in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the <jats:italic>ittA</jats:italic> mutant relative to its genetic parent, including <jats:italic>vraA, bba66</jats:italic>, <jats:italic>ospD</jats:italic> and <jats:italic>oms28</jats:italic> (<jats:italic>bba74</jats:italic>). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of <jats:italic>B. burgdorferi</jats:italic>.</jats:p><jats:sec><jats:title>AUTHOR SUMMARY</jats:title><jats:p>Lyme disease is a tick-borne infection mediated by the spirochetal bacterium, <jats:italic>Borrelia burgdorferi</jats:italic>, that is responsible for greater than 300,000 infections in the United States per year. As such, additional knowledge regarding how this pathogen modulates its regulatory armamentarium is needed to understand how <jats:italic>B. burgdorferi</jats:italic> establishes and maintains infection. The identification and characterization of small, non-coding RNA molecules in living systems, designated as sRNAs, has recalibrated how we view post-transcriptional regulation. Recently, over 1,000 sRNAs were identified in <jats:italic>B. burgdorferi</jats:italic>. Despite the identification of these sRNAs, we do not understand how they affect infectivity or <jats:italic>B. burgdorferi</jats:italic> pathogenesis related outcomes. Here, we characterize the <jats:italic>ittA B. burgdorferi</jats:italic> sRNA and show that it is essential for optimal infection using murine experimental infection as our readout. We also track the effect of this sRNA on the transcriptional and proteomic profile as the first step in providing mechanistic insight into how this important sRNA mediates its regulatory effect.</jats:p></jats:sec>

Transformation of Borrelia burgdorferi

“…It is possible that the promoter region of the downstream gene is within the coding region of the GOI and therefore replacement of GOI with resistance marker will impact the levels of the downstream gene. In addition, it has been recently shown that there is a likelihood of small RNA (sRNAs) and non‐coding RNA (ncRNAs) within the intergenic regions that can have localized or distal effects on gene expression profile impacting the overall phenotype observed with the transformants (Arnold et al., 2016; Arnold et al., 2018; Lybecker & Samuels, 2017; Medina‐Perez et al., 2020). These considerations should be factored into how the deletion construct is generated using suicide vectors as shown in Figures 1‐2.…”

An lp17-encoded small non-coding RNA with a potential regulatory role in mammalian host adaptation by the Lyme disease spirochete

“…In this study, we attempted to further characterize the bbd07/SR0726 locus of lp17 by determining whether the functional product encoded by this intergenic region is an RNA or protein, and assessing any potential regulatory effects resulting from its targeted investigators (44). We reasoned that a regulatory sRNA may have drastic effects on multiple bacterial characteristics if it were transcribed from a high-copy plasmid, and that investigation of these effects could provide insight into the native function of ittB.…”

Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogenB. burgdorferi

“…B. burgdorferi B31 gene annotations were downloaded from NCBI, RefSeq assembly accession GCF_000008685.2. sRNA annotations (Supplementary Table 2), as outlined above, were merged and sRNA gene names included for ssrA /tmRNA (SR0030), ssrS (6S) (SR0136) (Drecktrah et al, 2020), rnpB (SR0613), BsrW (SR0412) (Arnold et al, 2016), and IttA (SR0736) (Medina-Perez et al, 2020). The following sRNAs were missed from Supplementary Table 2 because of a lack of a 3′/5′ end within 75 nt of the previous sRNA annotations and were manually added: srp , ffs (4.5S) (SR0360), DsrA (SR0440) (Lybecker and Samuels, 2007) and IttB (Crowley and Bankhead, 2020).…”

“…B. burgdorferi RNA-mediated regulation has not been well characterized. Two putative base-pairing sRNAs have been suggested to have regulatory roles and/or impact infectivity (Lybecker and Samuels, 2007; Medina-Perez et al, 2020), but their direct base- pairing function has not been demonstrated. Further, no cis- acting RNAs have been described in B. burgdorferi .…”