PLoS ONE

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

Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones

Lyme borreliosis is the most important vector-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a Ge-LC-MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify up-regulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT-PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. This article is protected by copyright. All rights reserved

Identification of Borrelia protein candidates in mouse skin for potential diagnosis of disseminated Lyme borreliosis

Abstract In vector-borne diseases, the skin plays an essential role in the transmission of vector-borne pathogens between the vertebrate host and blood-feeding arthropods and in pathogen persistence. Borrelia burgdorferi sensu lato is a tick-borne bacterium that causes Lyme borreliosis (LB) in humans. This pathogen may establish a long-lasting infection in its natural vertebrate host where it can persist in the skin and some other organs. Using a mouse model, we demonstrate that Borrelia targets the skin regardless of the route of inoculation, and can persist there at low densities that are difficult to detect via qPCR, but that were infective for blood-feeding ticks. Application of immunosuppressive dermocorticoids at 40 days post-infection (PI) significantly enhanced the Borrelia population size in the mouse skin. We used non-targeted (Ge-LC-MS/MS) and targeted (SRM-MS) proteomics to detect several Borrelia-specific proteins in the mouse skin at 40 days PI. Detected Borrelia proteins included flagellin, VlsE and GAPDH. An important problem in LB is the lack of diagnosis methods capable of detecting active infection in humans suffering from disseminated LB. The identification of Borrelia proteins in skin biopsies may provide new approaches for assessing active infection in disseminated manifestations

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Proteomic analyses of <i>B</i>. <i>burgdorferi</i> ss, 297 parental strain and its clone 297/4.

<p>(A) The Venn diagram shows the protein overlap and the proteins specifically identified in the parental strain and the clone 297/4. (B) Graphic representation of the breakdown of the proteins specifically identified in the clone. Categorization was based upon JCVI annotation. The percentages represent the fraction of that category within the proteins. (C) Gene location of the specific proteins identified in the clone 297/4.</p

Inflammatory profiles of <i>B</i>. <i>burgdorferi</i> ss, parental strain and its clone 297/4.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least, three mice were analyzed for each time point.</p

Bacterial loads in the skin.

<p>The spirochetal burden in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>. Values represent relative expression + SD of three independent experiments. Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). (d: days).</p

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Inflammatory profiles of the different strains of <i>B</i>. <i>burgdorferi</i> ss.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least three mice were analyzed for each time point.</p