Signalling pathway controlling cupD fimbrial genes expression and role in Pseudomonas aeruginosa pathogenesis

自然界中大多数微生物以表面相关群落的形式存在，称为生物膜。生物膜被描述为“微生物之城”，其中细菌被结合在一起形成固体基质。有令人信服的证据表明，生物膜生活方式是微生物维持受保护生态位的有效手段。在人体内形成生物膜可导致慢性感染，这种感染可持续终生并导致宿主死亡。生物膜的稳定性和弹性是一个重要的医学和工业问题。生物膜已显示出对抗生素治疗的耐药性，并且难以通过免疫系统根除。生物膜的形成遵循一系列事件，从细菌最初附着在宿主组织上开始。最初的附着需要细菌表面黏附素，这种黏附素通常与被称为菌毛的长纤维有关。在感染过程中，细菌应该适应并形成生物膜以持续存在或更积极地定植宿主。对于细菌来说，这些选择在很大程度上是基于当地的环境因素，并通过改变基因表达而受到影响。例如，细菌是否决定停止运动并开始附着可能导致这些含有长纤维的粘附素的组装。为了检测环境信号，细菌进化出了由两个组成部分组成的调节装置，一个用于信号检测，一个用于影响基因表达，这两个组成部分被称为传感器和响应调节器，它们紧密相连。机会致病菌铜绿假单胞菌是一种自由生物体的原型，具有在广泛的真核宿主中引起疾病的能力。铜绿假单胞菌是造成人类一些最严重感染的原因，包括与宿主防御功能受损相关的新出现的医院感染。它是囊性纤维化和急性肺病患者严重肺部和上呼吸道感染的原因。铜绿假单胞菌具有许多负责上述菌毛组装的基因（杯状基因）。此外，这种细菌具有广泛的双组分系统，使这种有机体具有无处不在和多用途的特征。最后，铜绿假单胞菌表现出显著的毒力范围，从弱毒分离株到高毒广谱菌株。在本研究中，我们将研究双组分调控系统（Rcs/Pvr）对cupD毛膜基因表达的作用。我们之所以选择研究这些基因，是因为它们位于高毒力铜绿假单胞菌PA14菌株的致病性岛上。这些基因在实验室菌株铜绿假单胞菌PAO1的基因组中没有发现。致病性岛是由可移动的遗传因子组成的，它们在人类、动物和植物的细菌性病原体的毒力中起着关键作用。我们的研究将为复杂的调控网络、Pvr/Rcs系统和特定的cupD基因靶点提供原始、详细和完整的分子基础。这些系统在生物膜和毒力中的作用的表征将提高我们对铜绿假单胞菌信号传导和菌毛组装的基本认识，并将为寻找干扰铜绿假单胞菌发病机制的铅分子提供分子细节。

Most microbes in nature exist as surface associated communities called biofilms. Biofilms are described as a 'city of microbes' in which bacteria are hold together into a solid matrix. There is compelling evidence that the biofilm lifestyle is an efficient means for microorganisms to maintain a protected niche. Establishment of biofilms in humans leads to chronic infection that can persist for life and lead to host death. It is well described that stability and resilience of biofilms is a major medical and industrial problem. Biofilms have been shown to display resistance to antibiotic treatments and are recalcitrant to eradication via the immune system. The formation of the biofilm obeys to a series of events that start with initial attachment of the bacterium on the host tissue. The initial attachment requires bacterial surface adhesins that are frequently associated with long fibres called fimbriae. In the course of the infection process, the bacteria should adapt and either form a biofilm to persist or colonize the host more aggressively. For bacteria, these choices are based in large part on local environmental cues, and are effected through altered gene expression. For example whether the bacteria decide to stop motility and start attachment may result in the assembly of these long fibres-containing adhesins. In order to detect the environmental cues bacteria have evolved regulatory devices that are made of two-components, one for signal detection and one for affecting gene expression, both components, called sensor and response regulator being intimately linked. The opportunistic pathogen Pseudomonas aeruginosa is a prototype of free-living organism with the capacity to cause disease in a wide range of eukaryotic hosts. P. aeruginosa is responsible for some of the most serious infections in humans, including emerging nosocomial infections associated with impairment of host defences. It is responsible for severe lung and upper respiratory tract infections in patients with Cystic Fibrosis and acute pneumopathies. P. aeruginosa is equipped with numerous genes responsible for the assembly of the above-mentioned fimbriae (cup genes). Moreover, this bacterium has a wide repertoire of two-component systems that give this organism an ubiquitous and versatile character. Finally, P. aeruginosa displays a remarkable range of virulence, from weakly virulent isolates to highly virulent broad-spectrum strains. In the present study we will investigate the role of a two-component regulatory system (Rcs/Pvr) on expression of cupD fimbrial genes. We have chosen to study those genes, because they are located on the pathogenicity island of the highly virulent P. aeruginosa PA14 strain. These genes are otherwise not found in the genome of the laboratory strain P. aeruginosa PAO1. Pathogenicity islands are of mobile genetic elements, which play a pivotal role in the virulence of bacterial pathogens of humans, animals and plants. Our study will bring original, detailed and integrated molecular basis about a complex network of regulation, the Pvr/Rcs system, and the specific cupD gene target. The characterization of the role of these systems in biofilm and virulence will improve our basic knowledge of signalling and fimbriae assembly in P. aeruginosa and will provide molecular details for the search of lead molecules interfering with P. aeruginosa pathogenesis.