This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Coxiella burnetii is an obligate intracellular pathogen that causes a flu-like syndrome in humans (Q fever) and abortions in livestock. The dormant 'spore-like'small cell variant (SCV) of C. burnetii is infectious (aerosol) and extremely resistant to environmental stressors (UV light, desiccation). Once inside an acidic (pH ~5) phagolysosome-like compartment of a eukaryotic cell, C. burnetii transforms into a vegetative large cell variant (LCV). Identification of dominant outer membrane proteins (OMPs) synthesized by both morphological forms would result in a better understanding of C. burnetii pathogenesis. To this end, OMPs of purified C. burnetii SCVs and LCVs were prepared (sarkosyl fractionation), separated (isoelectric focusing and SDS-PAGE), visualized (silver staining), and fingerprinted (mass spectrometry). Two dominant SCV-specific OMPs were identified as CBU0307 and CBU0311. BLASTO searches of NCBI clusters of orthologous groups (COG) database show that CBU0307 is orthologous to ompA-like outer membrane proteins and CBU0311 is a member of COG3637, opacity protein and related surface antigens. C. burnetii CBU0311 has been the focus of several previous studies and has been termed P1. Further in silico analyses identified 3 additional CBU0307 paralogues (CBU1260, CBU1600, and CBU1814) and 3 additional CBU0311 paralogues (CBU1412, CBU1413 and CBU1414) within the C. burnetii genome. We used quantitative RT-PCR to show that transcript levels of the p1-group [CBU0311 (here termed p1-A), p1-B (CBU1414), p1-C (CBU1413), and p1-D (CBU1412)] are highest at four days post-infection, suggesting that the corresponding proteins are synthesized as the bacterium transitions to the SCV. As orthologues of the p1-group have been shown to serve as adhesins for extracellular matrix (ECM) components, we purified recombinant P1-A and analyzed its ability to bind immobilized ECM components by ELISA. A statistically significant interaction with collagen was observed. Identification of SCV OMP's involved in host cell adherence could provide a frontline defense strategy against C. burnetii infections.
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