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HtrA serine proteases are highly conserved and essential ATP-independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN-PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn(2+) Cu(2+)Mn(2+). Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl2 at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA(S226A)). Although inoculation with either BbHtrA or BbHtrA(S226A) produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.

A basic obligation of a commercial clinical laboratory is to validate a test before offering it for sale to diagnose patients. Advanced Laboratory Services offers a culture method to diagnose patients with a Borrelia species infection ([1][1]), but we do not find the evidence provided in support of

A novel method of culturing spirochetes from the serum of U.S. Lyme disease patients was recently reported by Sapi and colleagues to have 94% sensitivity and 100% specificity for Borrelia species as assessed by microscopy and DNA sequence analysis of the pyrG gene (E. Sapi, N. Pabbati, A. Datar, E. M. Davies, A. Rattelle, and B. A. Kuo, Int. J. Med. Sci. 10:362–376, 2013). The majority of the spirochetes described were related by pyrG sequences to species of Borrelia previously undetected in North American patients without a reported history of travel to Europe or Asia. To better understand these unexpected findings, we determined pyrG sequences of the laboratory reference strains used by the investigators for method development and testing of culture medium. Eighty percent (41/51) of the reported patient-derived pyrG sequences were identical to one of the laboratory strains, and an additional 12% (6/51) differed by only a single nucleotide across a 603-bp region of the pyrG gene. Thus, false positivity due to laboratory contamination of patient samples cannot be ruled out, and further validation of the proposed novel culture method is required.

Summary The Lyme disease spirochaete, Borrelia burgdorferi, causes damage to diverse host tissues and induces inflammation but the mechanisms of injury are poorly understood. We recently reported that a surface-exposed B. burgdorferi protease, which is expressed during human disease and is conserved within the major Lyme disease spirochaete species, degrades the extracellular matrix proteoglycan, aggrecan. Here we demonstrate that BbHtrA also degrades fibronectin and numerous proteoglycans found in skin, joints and neural tissues. BbHtrA degradation of fibronectin released known pro-inflammatory fibronectin fragments FnIII13–14 and Fnf-29, which may amplify the inflammatory processes triggered by the presence of the bacteria. When this hypothesis was tested directly by exposing chondrocytes to BbHtrA in vitro, inflammatory cytokines (sICAM-1 and IL-6) and chemokines (CXCL1, CCL1, CCL2 and CCL5) that are hallmarks of Lyme disease were induced. These results provide the first evidence that, by utilizing BbHtrA, B. burgdorferi may actively participate in its dissemination and in the tissue damage and inflammation observed in Lyme disease.

Connective tissues are the most common area of colonization for the Lyme disease spirochaete Borrelia burgdorferi. Colonization is aided by the interaction between numerous bacterial adhesins with components of the extracellular matrix (ECM). Here we describe a novel interaction between B. burgdorferi and the major ECM proteoglycan found in joints, aggrecan. Using affinity chromatography and mass spectrometry we identify two borrelial aggrecan-binding proteins: the known ECM ligand Bgp (BB0588) and an uncharacterized protease BbHtrA (BB0104). Proteinase K studies demonstrate that BbHtrA is surface exposed. Immunoblots using sera from patients with both early and late Lyme disease establish that BbHtrA is expressed during human disease, immunogenic, and conserved in the three major Lyme disease spirochaete species. Consequences of the interaction between aggrecan and BbHtrA were examined by proteolysis assays. BbHtrA cleaves aggrecan at a site known to destroy aggrecan function and which has been previously observed in the synovial fluid of patients with Lyme arthritis. These data demonstrate that B. burgdorferi possess aggrecan-binding proteins which may provide the organism with additional capability to colonize connective tissues. Moreover, our studies provide the first evidence that B. burgdorferi possess proteolytic activity which may contribute to the pathogenesis of Lyme arthritis.