Pathogenesis of Haemophilus Ducreyi Infections

描述（由申请人提供）：杜氏嗜血杆菌是一种严格意义上的人类病原体，可导致软下疳，一种生殖器溃疡疾病（GUD），促进人类免疫缺陷病毒（HIV-1）的传播。为了研究杜氏嗜血杆菌的生物学，我们建立了一个接近自然感染的人体感染模型。在感染期间，杜氏嗜血杆菌在脓肿的敌对环境中被发现并抵抗吞噬作用。我们发现H. ducreyi对宿主有广泛的转录反应，这表明H. ducreyi对宿主环境有感知和响应。H. ducreyi只包含两个已知的对胞质外应激作出反应的系统：2组分调节因子CpxRA和替代sigma因子RpoE。一些CpxRA特异性效应物的同源物在脓包中被上调，而许多RpoE特异性效应物的同源物在脓包中被下调，这表明CpxRA和RpoE系统在H. ducreyi中联系在一起，并以协调的方式对宿主施加的胁迫作出反应。我们发现CpxRA控制了H. ducreyi几个主要毒力决定因素的表达，CpxRA不受控制的激活会损害该生物感染人类志愿者的能力。我们假设H. ducreyi通过CpxRA和RpoE感知宿主环境，CpxRA和RpoE在感染期间都发挥作用，以对抗体内遇到的压力并控制毒力决定因子的产生，CpxRA或RpoE系统中的组成活性表达或缺失突变体将因毒力而减弱，并且已知或新的毒力决定因子将在感染期间受到CpxRA和RpoE的差异调节。为了验证这些假设，我们的具体目标包括：1)评估cpxR、rpoE、rseA和rseC缺失突变体以及表达组成型激活cpxR和rpoE的菌株对人类志愿者的毒力；2)确定CpxRA和RpoE系统对人类感染相关模型发病机制的作用机制；3)鉴定由CpxRA和RpoE途径控制的新型毒力决定因素，并评估它们在体内（ai1）和体外（ai2）模型感染中的作用。我们的建议提供了一个独特的机会来研究两个相互关联的应激反应系统对人类病原体生存的贡献，将导致新的毒力决定因素的鉴定，并已经导致了一种新的抗菌策略的发展。经NIAID批准，本次修订的目的是获得资金支持与资助申请的目标1和目标3相关的人接种实验。

DESCRIPTION (provided by applicant): Haemophilus ducreyi is a strict human pathogen that causes chancroid, a genital ulcer disease (GUD) that facilitates the transmission of the human immunodeficiency virus (HIV-1). To study the biology of H. ducreyi, we developed a human challenge model that closely simulates natural infection. During infection, H. ducreyi is found in the hostile environment of an abscess and resists phagocytosis. We found that H. ducreyi had a broad transcriptional response to the host, suggesting that H. ducreyi senses and responds to the host environment. H. ducreyi contains only two systems known to respond to extracytoplasmic stress: the 2- component regulator CpxRA and the alternative sigma factor, RpoE. Several H. ducreyi homologues of CpxRA-specific effectors were upregulated, while many homologues of RpoE-specific effectors were downregulated in pustules, suggesting that that CpxRA and RpoE systems are linked in H. ducreyi and function in a coordinated fashion to respond to stresses mounted by the host. We found that CpxRA controlled the expression of several major virulence determinants of H. ducreyi and that uncontrolled activation of CpxRA impaired the ability of the organism to infect human volunteers. We hypothesize that H. ducreyi senses the host environment via CpxRA and RpoE, that both CpxRA and RpoE function during infection to combat stresses encountered in vivo and control the production of virulence determinants, that constitutively active expressers or deletion mutants in the CpxRA or RpoE systems will be attenuated for virulence and that known or novel virulence determinants will be differentially regulated by CpxRA and RpoE during infection. To test these hypotheses, our specific aims include: 1) evaluation of cpxR, rpoE, rseA and rseC deletion mutants and strains that express constitutively activated CpxR and RpoE for virulence in human volunteers; 2) determination of the mechanism(s) underlying the contributions of the CpxRA and RpoE systems to pathogenesis in models relevant to human infection; 3) identification of novel virulence determinants controlled by the CpxRA and RpoE pathways and evaluation of their role in infection in the in vivo (Aim1) and in vitro (Aim 2) models. Our proposal offers the unique opportunity to study the contributions of two interrelated stress response systems to the survival of a pathogen in humans, will lead to the identification of novel virulence determinants, and has already led to the development of a novel antimicrobial strategy. As approved by NIAID, the purpose of this revision is to obtain funds to support the human inoculation experiments related to Aims 1 and 3 of the funded application. PUBLIC HEALTH RELEVANCE: Haemophilus ducreyi is a bacterium that causes a genital ulcer disease that facilitates HIV transmission. H. ducreyi turns on the expression of many of its genes when it infects the human host. Our study is designed to examine how H. ducreyi uses stress response systems to adapt to the human host. H. ducreyi is only killed by three classes of antibiotics and is a high priority organism for the development of new therapies. This project should yield targets of vaccines or alternative treatments for H. ducreyi and other bacterial infections.