Heterogeneity of Borrelia burgdorferi Sensu Stricto Population and Its Involvement in Borrelia Pathogenicity: Study on Murine Model with Specific Emphasis on the Skin Interface

RESEARCH ARTICLE Heterogeneity of Borrelia burgdorferi Sensu Stricto Population and Its Involvement in Borrelia Pathogenicity: Study on Murine Model with Specific Emphasis on the Skin Interface Aurélie Kern1,3, Gilles Schnell2, Quentin Bernard1, Amandine Bœuf2, Benoît Jaulhac1, Elody Collin1, Cathy Barthel1, Laurence Ehret-Sabatier2☯, Nathalie Boulanger1☯* 1 EA7290: Virulence bactérienne précoce: groupe Borréliose de Lyme, Facultés de médecine et de pharmacie, Université de Strasbourg, Strasbourg, France, 2 Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, Université de Strasbourg, CNRS, UMR7178, Strasbourg, France, 3 Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts, United States of America OPEN ACCESS Citation: Kern A, Schnell G, Bernard Q, Bœuf A, Jaulhac B, Collin E, et al. (2015) Heterogeneity of Borrelia burgdorferi Sensu Stricto Population and Its Involvement in Borrelia Pathogenicity: Study on Murine Model with Specific Emphasis on the Skin Interface. PLoS ONE 10(7): e0133195. doi:10.1371/ journal.pone.0133195 Editor: Brian Stevenson, University of Kentucky College of Medicine, UNITED STATES Received: November 10, 2014 Accepted: June 23, 2015 Published: July 21, 2015 Copyright: © 2015 Kern 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Aurélie Kern was supported by a grant #2009.60.053 from the Conseil Régional d’Alsace and Direction Générale de l'Armement. Quentin Bernard is supported by a grant 2012.60.0033 from the Conseil Régional d’Alsace and Direction Générale de l'Armement. Nathalie Boulanger was supported by a Fulbright grant and a Monahan grant for her sabbatical. ☯ These authors contributed equally to this work. * Abstract Lyme disease is a multisystemic disorder caused by B. burgdorferi sl. The molecular basis for specific organ involvement is poorly understood. The skin plays a central role in the development of Lyme disease as the entry site of B. burgdorferi in which specific clones are selected before dissemination. We compared the skin inflammatory response (antimicrobial peptides, cytokines and chemokines) elicited by spirochete populations recovered from patients presenting different clinical manifestations. Remarkably, these spirochete populations induced different inflammatory profiles in the skin of C3H/HeN mice. As spirochete population transmitted into the host skin is heterogeneous, we isolated one bacterial clone from a population recovered from a patient with neuroborreliosis and compared its virulence to the parental population. This clone elicited a strong cutaneous inflammatory response characterized by MCP-1, IL-6 and antimicrobial peptides induction. Mass spectrometry of this clone revealed 110 overexpressed proteins when compared with the parental population. We further focused on the expression of nine bacterial surface proteins. bb0347 coding for a protein that interacts with host fibronectin, allowing bacterial adhesion to vascular endothelium and extracellular matrix, was found to be induced in host skin with another gene bb0213 coding for a hypothetical protein. These findings demonstrate the heterogeneity of the B. burgdorferi ss population and the complexity of the interaction involved early in the skin. PLOS ONE | DOI:10.1371/journal.pone.0133195 July 21, 2015 1 / 16 Skin and Borrelia Transmission Competing Interests: The authors have declared that no competing interests exist. Introduction Lyme disease, a zoonosis caused by the Borrelia burgdorferi sensu lato (sl) group is the most common arthropod-borne disease in the Northern Hemisphere. Among the different B. burgdorferi sl species, B. burgdorferi ss sensu stricto (ss) is a pathogenic species present in both the United States and Europe. Lyme disease induces a multisystemic disorder typically characterized first by a skin inflammation, the erythema migrans at the site of Borrelia inoculation in human. After hematogenous dissemination systemic manifestations (eg, cardiac, articular, neurological and cutaneous) are observed [1]. The factors responsible for Borrelia organotropism are not known. In a mouse model, the skin has been shown to constitute a key interface where the spirochetes multiply before disseminating to target organs [2]. To better evaluate the role of the vertebrate host skin in the transmission, we studied different B. burgdorferi isolates recovered from patients with distinct clinical manifestations. The skin protects itself against infections by innate immunity and is important to maintain homeostasis [3]. Recently the role of the skin has been re-evaluated in Lyme borreliosis with specific emphasis on the role of OspC in the transmission [4–6] and as a potential filter in B. burgdorferi populations [7]. We selected genes of innate immunity (antimicrobial peptides, chemokines and cytokines) and compared the skin inflammatory response elicited by human isolates of B. burgdorferi in a mouse model, as the immune pressure in the skin might drive certain Borrelia populations towards specific organs. Spirochetes refer to distinct species and genomic groups and differ in their pathogenicity [8]. Two main genetic markers, the outer-surface protein C (ospC) type and the 16S-23S rRNA intergenic spacer type (RST) can be used to distinguish strains of B. burgdorferi ss allowing the strains to correlate the severity of their clinical manifestations [9]. Three RSTs have been defined: RST1 genotype is correlated with hematogenous dissemination and severe clinical signs, RST3 isolates are considered as non-disseminating strains, and RST2 isolates as intermediate. Within the former marker, 21 OspC groups have been initially defined [10]. In patients, hematogenous dissemination is mainly associated with types A, B, I and H among the 16 actual OspC genotypes [11]. These genetic markers are currently the best to define specific clinical isolates of B. burgdorferi [12]. However, they are not sufficient to determine the outcome of an infection initiated by a specific Borrelia strain and species. The situation is complicated because of the biodiversity of B. burgdorferi strains in ticks and tissues [7] and the differing disseminating profiles in clones isolated from one specific Borrelia population [13]. Clearly, a selection occurs but the factors that govern it remain to be identified. In this study we compared the tissue distribution and the inflammatory response of different clinical isolates (also referred to as pathotypes) belonging to different RST groups. Considering that the skin could act as a filter to select specific clones, we selected a specific clo (...truncated)