HrpA, an RNA Helicase Involved in RNA Processing, Is Required for Mouse Infectivity and Tick Transmission of the Lyme Disease Spirochete Aydan Salman-Dilgimen 1 , Pierre-Olivier Hardy 1 , Justin D. Radolf 2,3,4,5,6 , Melissa J. Caimano 2,3,4 , George Chaconas 1 * 1 Department of Biochemistry & Molecular Biology and Department of Microbiology, Immunology & Infectious Diseases, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada, 2 Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 3 Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 4 Department of Molecular Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 5 Department of Genetics & Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 6 Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, United States of America Abstract The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete’s global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 [p66], bba74, bb0241 [glpK], bb0242 and bb0243 [glpA]) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete’s enzootic cycle. Citation: Salman-Dilgimen A, Hardy P-O, Radolf JD, Caimano MJ, Chaconas G (2013) HrpA, an RNA Helicase Involved in RNA Processing, Is Required for Mouse Infectivity and Tick Transmission of the Lyme Disease Spirochete. PLoS Pathog 9(12): e1003841. doi:10.1371/journal.ppat.1003841 Editor: D. Scott Samuels, The University of Montana, United States of America Received July 15, 2013; Accepted November 4, 2013; Published December 19, 2013 Copyright: ß 2013 Salman-Dilgimen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH/NIAID grants AI29735 (MJC and JR) and AI85248 (MJC), and grant MOP 53086 (GC) from the Canadian Institutes of Health Research (http://www.cihr-irsc.gc.ca/e/193.html). GC also holds a Canada Research Chair in the Molecular Biology of Lyme Borreliosis (http://www.chairs- chaires.gc.ca/home-accueil-eng.aspx) and a Scientist Award from Alberta Innovates – Health Soultions (http://www.ahfmr.ab.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chaconas@ucalgary.ca Introduction Lyme borreliosis is the most prevalent vector-transmitted disease in the northern hemisphere and has a significant impact on human health (see [1,2]). The disease is caused by the spirochete Borrelia burgdorferi and related species. B. burgdorferi is maintained in nature by a complex enzootic cycle that involves ticks as vectors and vertebrate animals as reservoir hosts. Survival in the very disparate arthropod and animal environments demands changes in the expression of numerous genes in a precise manner [1,3,4]. The primary global regulators for these differentially-expressed genes are the alternative sigma factors RpoN and RpoS, which substitute for RpoD (s 70 ) in the RNA polymerase holoenzyme to effect transcription in response to environmental signals perceived during tick transmission and the mammalian phase of the enzootic cycle [5,6,7]. Other players in the RpoN-RpoS pathway are the response regulatory protein Rrp2 [8,9,10,11] and the Fur/PerR ortholog, BosR [12,13]. In addition to the control of gene expression at the transcrip- tional level, RNA-mediated regulation has emerged as a burgeoning field [14,15,16,17,18,19]. Little is known regarding RNA regulation in B. burgdorferi. However, a small RNA (DsrA) [20] along with the RNA chaperone Hfq [21] and the RNA binding protein CsrA [22,23] have been shown to regulate expression of rpoS/RpoS. In the expanding world of RNA regulation, RNA helicases have emerged as major players. RNA helicases, universal enzymes known to play roles in all cellular processes involved in RNA metabolism [24,25,26,27,28,29], also have a connection to a number of infectious diseases [30]. RNA helicases are highly conserved enzymes that unwind double-stranded RNA in an ATP- dependent manner. RNA helicases are categorized into families PLOS Pathogens | www.plospathogens.org 1 December 2013 | Volume 9 | Issue 12 | e1003841