Bordetella Biofilms and Pathogenesis

描述（由申请人提供）：百日咳，由百日咳博德泰拉引起的百日咳在美国仍然是一个相当大的经济和健康负担，尽管疫苗接种覆盖率很高。目前的百日咳疫苗虽然能有效预防严重形式的疾病，但不能预防携带者状态。成人和青少年携带者负责将细菌传播给婴儿和幼儿，对婴儿和幼儿来说，这种疾病是严重的，有时是致命的。迫切需要确定能够防止细菌携带的新因素，并将其纳入无细胞疫苗。据推测，人类的鼻咽携带百日咳是由于它能够以一种被称为生物膜的无底形式存在。我们的初步结果表明，百日咳在小鼠中以粘附在鼻上皮上的多细胞群落存在，这让人想起生物膜。本研究的总体目标是研究生物膜相关Bps多糖在生物膜发育和发病机制中的作用，并深入了解生物膜和呼吸道中控制Bps表达的调控通路。Bps类似于其他细菌（包括许多病原体）的PGA/PIA/PNAG（聚-2-1,6-N-乙酰氨基葡萄糖胺）多糖。在Specific Aim 1中，生物学相关的体外模型系统将被用来研究Bps在生物膜形成中的作用的机制基础。Bps在提供抗微生物化合物保护方面的重要性将通过测试其在授予对抗生素、抗菌肽和活性氧的抗性方面的作用来检验。据推测，Bps通过发挥免疫调节作用和靶向免疫系统的一种或多种成分，介导了百日咳在呼吸道中的存活和持续。百日咳鼻内感染小鼠模型将被用来检验这些假设。在Specific Aim 2中，bps在生物膜形成过程和呼吸道中的时空表达梯度将通过实时RT - PCR检测和GFP荧光测量来检测。最后，为了深入了解控制bps表达的复杂网络，我们将研究BpsR调节蛋白在生物膜和小鼠呼吸道中控制bps位点表达的作用。与公共卫生有关。本研究将有助于进一步了解百日咳在哺乳动物宿主中的致病机制和感染周期。我们的研究将刺激以bps为基础的疫苗和百日咳抗体等试剂的开发。由于bps样多糖是由葡萄球菌、大肠杆菌和耶尔森氏菌等多种病原体产生的，因此我们的研究结果将有助于治疗和了解各种细菌感染。

DESCRIPTION (provided by applicant): Pertussis, caused by Bordetella pertussis remains a considerable economic and health burden in the USA, despite very high coverage of vaccination. Current pertussis vaccines although effective at preventing the severe form of disease do not prevent the carrier state. Adult and adolescent carriers are responsible for the transmission of the bacterium to infants and young children, in whom the disease is severe and sometimes lethal. There is an urgent need to identify new factors that will be protective against bacterial carriage and will be included in the acellular vaccine. It is hypothesized that nasopharyngeal carriage of B. pertussis in humans is due to its ability to exist in a sessile form known as biofilms. Our preliminary results demonstrate that B. pertussis exists in mice as multicellular community adherent to the nasal epithelium, which are reminiscent of biofilms. The overall objectives of this research are to study the role of the biofilm-associated Bps polysaccharide in biofilm development and pathogenesis and to gain insight into the regulatory circuitry that controls bps expression in biofilms and in the respiratory tract. Bps is similar to PGA/PIA/PNAG (poly-2-1,6-N- acetylglucosamine) polysaccharides of other bacteria including a number of pathogens. In Specific Aim 1, biologically relevant in vitro model systems will be utilized to study the mechanistic basis of the role of Bps in biofilm formation. The importance of Bps in providing protection from anti-microbial compounds will be examined by testing its role in conferring resistance against antibiotics, antimicrobial peptides and reactive oxygen species. It is hypothesized that Bps mediates survival and persistence of B. pertussis in the respiratory tract by playing an immunomodulatory role and by targeting one or more components of the immune system. Mouse models of B. pertussis intranasal infection will be employed to test these hypotheses. In Specific Aim 2, the establishment of temporal and spatial expression gradient of bps during biofilm formation and in the respiratory tract will be examined by real time RT PCR assays and measurement of GFP fluorescence. Finally, to provide insights into the complex networks that operate to control bps expression, the role of a regulatory protein BpsR in controlling the expression of the bps locus in biofilms and in the mouse respiratory tract will be examined. Relevance to public health. This project will provide a better understanding of the pathogenic mechanisms and the infectious cycle of B. pertussis in mammalian hosts. Our studies will stimulate the development of Bps-based vaccines and reagents like antibodies for treatment of pertussis. Since Bps-like polysaccharides are produced by a number of pathogens like Staphylococci, E. coli and Yersinia Spp, and possibly others, our results will contribute towards the treatment and the understanding of a wide variety of bacterial infections.