Haemophilus Hap-mediated Microcolony Formation

描述（由申请人提供）：不可分型的流感嗜血杆菌是局部呼吸道疾病的常见病因，包括中耳炎、鼻窦炎、支气管炎和肺炎。此外，这种微生物引起严重的全身性疾病，如脑膜炎、心内膜炎和败血症。不可分型流感嗜血杆菌疾病发病机制的第一步涉及上呼吸道粘膜的定植。我们已经确定了一种名为Hap的流感嗜血杆菌丝氨酸蛋白酶，它促进了与上皮细胞和细胞外基质蛋白的密切相互作用，也促进了细菌聚集和微集落的形成。根据我们的体外实验结果，我们推测Hap在定植过程中起重要作用。Hap属于不断增长的自转运蛋白家族，作为具有3个功能域的前体蛋白合成，包括n端信号序列，具有粘附活性的内部蛋白酶结构域（Haps）和具有转运活性的c端外膜结构域（HapBeta）。最终，Hap经历自身蛋白水解裂解，并在细胞外释放Hap。在最近的研究中，我们证明了一种被称为分泌性白细胞蛋白酶抑制剂（SLPI）的宿主蛋白增强了Hap介导的粘附和微集落形成。这种蛋白存在于呼吸道分泌物中，抑制Hap自身蛋白水解，导致表面相关Hap的积累。在目前的建议中，我们将重点关注hap介导的粘附和微菌落形成。在目标1中，我们将求解Haps的晶体结构，并定义参与粘附和微菌落形成的相互作用表面。在Aim 2中，我们将在栗鼠中耳炎模型中研究微菌落抵抗阳离子肽杀伤、逃避巨噬细胞吞噬和增强持久性的能力。在Aim 3中，我们将描述呼吸道病毒感染与hap介导的粘附和微集落形成之间的关系，重点关注SLPI的作用。从实际的角度来看，拟议的研究可能有助于制定治疗和预防流感嗜血杆菌疾病的新策略。也许更重要的是，它们可以提供对宿主-微生物关系的一般见解，并扩展我们对微生物生物膜的理解。

DESCRIPTION (provided by applicant): Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, including otitis media, sinusitis, bronchitis, and pneumonia. In addition, this organism causes serious systemic disease, such as meningitis, endocarditis, and septicemia. The initial step in the pathogenesis of nontypable H. influenzae disease involves colonization of the upper respiratory mucosa. We have identified an H. influenzae serine protease called Hap, which facilitates intimate interaction with epithelial cells and extracellular matrix proteins and also promotes bacterial aggregation and microcolony formation. Based on our in vitro results, we speculate that Hap plays an important role in the process of colonization. Hap belongs to the growing family of autotransporter proteins and is synthesized as a precursor protein with 3 functional domains, including an N-terminal signal sequence, an internal protease domain with adhesive activity (Haps), and a C-terminal outer membrane domain with translocator activity (HapBeta). Ultimately, Hap undergoes autoproteolytic cleavage, with extracellular release of Haps. In recent work, we demonstrated that Hap mediated adherence and microcolony formation are potentiated by a host protein called secretory leukocyteprotease inhibitor (SLPI). This protein is present in respiratory secretions and inhibits Hap autoproteolysis, resulting in accumulation of surface-associated Haps. In the present proposal, we will focus on Hap-mediated adherence and microcolony formation. In Aim 1, we will solve the crystal structure of Haps and define the interactive surfaces involved in adherence and microcolony formation. In Aim 2, we will examine the ability of microcolonies to resist killing by cationic peptides, to evade macrophage phagocytosis, and to enhance persistence in the chinchilla otitis media model. In Aim 3, we will characterize the relationship between respiratory viral infection and Hap-mediated adherence and microcolony formation, concentrating on the role of SLPI. From a practical perspective, the proposed studies may facilitate efforts to develop novel strategies forthe treatment and prevention of H. influenzae disease. Perhaps more importantly, they may provide general insights into host-microbe relationships and expand our understanding of microbial biofilms.