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Abstract：
Similar to Gram-negative organisms, Borrelia spirochetes are dual-membrane organisms with both an inner and outer membrane. Although the outer membrane contains integral membrane proteins, few of the borrelial outer membrane proteins (OMPs) have been identified and characterized to date. Therefore, we utilized a consensus computational network analysis to identify novel borrelial OMPs.
Using a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form β-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes.
Using a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.
Using a series of computer-based algorithms, we selected all protein-encoding sequences predicted to be OM-localized and/or to form β-barrels in the borrelial OM. Using this system, we identified 41 potential OMPs from B. burgdorferi and characterized three (BB0838, BB0405, and BB0406) to confirm that our computer-based methodology did, in fact, identify borrelial OMPs. Triton X-114 phase partitioning revealed that BB0838 is found in the detergent phase, which would be expected of a membrane protein. Proteolysis assays indicate that BB0838 is partially sensitive to both proteinase K and trypsin, further indicating that BB0838 is surface-exposed. Consistent with a prior study, we also confirmed that BB0405 is surface-exposed and associates with the borrelial OM. Furthermore, we have shown that BB0406, the product of a co-transcribed downstream gene, also encodes a novel, previously uncharacterized borrelial OMP. Interestingly, while BB0406 has several physicochemical properties consistent with it being an OMP, it was found to be resistant to surface proteolysis. Consistent with BB0405 and BB0406 being OMPs, both were found to be capable of incorporating into liposomes and exhibit pore-forming activity, suggesting that both proteins are porins. Lastly, we expanded our computational analysis to identify OMPs from other borrelial organisms, including both Lyme disease and relapsing fever spirochetes.
Using a consensus computer algorithm, we generated a list of candidate OMPs for both Lyme disease and relapsing fever spirochetes and determined that three of the predicted B. burgdorferi proteins identified were indeed novel borrelial OMPs. The combined studies have identified putative spirochetal OMPs that can now be examined for their roles in virulence, physiology, and disease pathogenesis. Importantly, the studies described in this report provide a framework by which OMPs from any human pathogen with a diderm ultrastructure could be cataloged to identify novel virulence factors and vaccine candidates.
摘要：
类似于革兰氏阴性菌，螺旋体是具有内膜和外膜的双膜生物。虽然外膜含有完整的膜蛋白，迄今为止，很少有疏螺旋体外膜蛋白（OMPs）被鉴定和表征。因此,我们利用共识计算网络分析来识别新的疏螺旋体OMP。
使用一系列基于计算机的算法，我们选择了所有预测为OM定位和/或在疏螺旋体OM中形成β桶的蛋白质编码序列。使用这个系统，我们从B.burgdorferi鉴定了41个潜在的OMP，并表征了三个（BB0838，BB0405和BB0406），以确认我们基于计算机的方法确实如此，事实上,确定疏螺旋体OMP。TritonX-114相分配表明在洗涤剂相中发现BB0838，这是膜蛋白的预期。蛋白水解试验表明BB0838对蛋白酶K和胰蛋白酶均部分敏感,进一步表明BB0838是表面暴露的。与先前的研究一致，我们还确认BB0405是表面暴露的，并且与交界性OM相关。此外，我们已经证明BB0406，一个共同转录的下游基因的产物，还编码了一部小说，以前未表征的疏螺旋体OMP。有趣的是,虽然BB0406具有与OMP一致的几种物理化学性质，发现它对表面蛋白水解具有抗性。与BB0405和BB0406为OMP一致，发现两者都能够掺入脂质体并表现出孔形成活性，这表明这两种蛋白质都是孔蛋白。最后,我们扩展了我们的计算分析，以识别来自其他疏螺旋体生物的OMPs，包括莱姆病和复发性发热螺旋体。
使用一致的计算机算法，我们生成了莱姆病和复发性发热螺旋体的候选OMPs列表,并确定了3种预测的伯氏螺旋体B.burgdorferi蛋白确实是新型的疏螺旋体OMPs.联合研究已经确定了推定的螺旋体OMPs，现在可以检查它们在毒力中的作用，生理学,和疾病的发病机理。重要的是,本报告中描述的研究提供了一个框架,通过该框架,可以对任何具有表皮超微结构的人类病原体的OMPs进行分类,以鉴定新的毒力因子和候选疫苗.
使用一系列基于计算机的算法，我们选择了所有预测为OM定位和/或在疏螺旋体OM中形成β桶的蛋白质编码序列。使用这个系统，我们从B.burgdorferi鉴定了41个潜在的OMP，并表征了三个（BB0838，BB0405和BB0406），以确认我们基于计算机的方法确实如此，事实上,确定疏螺旋体OMP。TritonX-114相分配表明在洗涤剂相中发现BB0838，这是膜蛋白的预期。蛋白水解试验表明BB0838对蛋白酶K和胰蛋白酶均部分敏感,进一步表明BB0838是表面暴露的。与先前的研究一致，我们还确认BB0405是表面暴露的，并且与交界性OM相关。此外，我们已经证明BB0406，一个共同转录的下游基因的产物，还编码了一部小说，以前未表征的疏螺旋体OMP。有趣的是,虽然BB0406具有与OMP一致的几种物理化学性质，发现它对表面蛋白水解具有抗性。与BB0405和BB0406为OMP一致，发现两者都能够掺入脂质体并表现出孔形成活性，这表明这两种蛋白质都是孔蛋白。最后,我们扩展了我们的计算分析，以识别来自其他疏螺旋体生物的OMPs，包括莱姆病和复发性发热螺旋体。
使用一致的计算机算法，我们生成了莱姆病和复发性发热螺旋体的候选OMPs列表,并确定了3种预测的伯氏螺旋体B.burgdorferi蛋白确实是新型的疏螺旋体OMPs.联合研究已经确定了推定的螺旋体OMPs，现在可以检查它们在毒力中的作用，生理学,和疾病的发病机理。重要的是,本报告中描述的研究提供了一个框架,通过该框架,可以对任何具有表皮超微结构的人类病原体的OMPs进行分类,以鉴定新的毒力因子和候选疫苗.
