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

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.

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

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.

Tick saliva represses innate immunity and cutaneous inflammation in a murine model of Lyme disease.

Lyme borreliosis is an arthropod-borne disease transmitted by the Ixodes tick. This spirochetal infection is first characterized by a local cutaneous inflammation, the erythema migrans. The skin constitutes a key interface in the development of the disease. During Borrelia inoculation, tick saliva affects the innate and adaptive immunity of the vertebrate host skin. Some key mediators of innate immunity such as antimicrobial peptides (cathelicidin and defensin families) have been identified as important initiators of skin inflammation. We analyzed the role of tick saliva on integumental innate immunity using different protocols of Borrelia infection, via syringe or direct tick transmission. When syringe inoculation was used, Borrelia triggered skin inflammation with induction of CRAMP, the mouse cathelicidin, and tumor necrosis factor-alpha. However, when Borrelia was transmitted directly via the tick, we observed a significant repression of inflammatory genes, suggesting a critical role of tick saliva in skin innate immunity. For all the protocols tested, a peak of intense Borrelia multiplication occurred in the skin between days 5 and 15, before bacterial dissemination to target organs. We conclude that Borrelia pathogens specifically use the tick saliva to facilitate their transmission to the host and that the skin constitutes an essential interface in the development of Lyme disease.