Haemophilus Hap-mediated Microcolony Formation

非典型流感嗜血杆菌是局部呼吸道疾病的常见原因， 包括中耳炎、鼻窦炎、支气管炎和肺炎。此外，这种微生物会引起严重的 全身性疾病，如脑膜炎、心内膜炎和败血症。发病机制的最初步骤 不典型的H.流感疾病涉及上呼吸道粘膜的定植。我们已经确定 一个H.流感病毒丝氨酸蛋白酶称为Hap，其促进与上皮细胞的密切相互作用， 细胞外基质蛋白，并且还促进细菌聚集和小菌落形成。基于 结合我们的体外实验结果，我们推测Hap在其定植过程中起着重要的作用。哈普属于 属于不断增长的自转运蛋白家族，并作为具有3个功能的前体蛋白合成 结构域，包括N-末端信号序列，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-末端外膜结构域（HapB）。最后哈普经历了 自身蛋白水解裂解，伴随Haps的细胞外释放。在最近的工作中，我们证明了Hap- 一种称为分泌性白细胞的宿主蛋白质可增强介导的粘附和小菌落形成 蛋白酶抑制剂（SLPI）。这种蛋白质存在于呼吸道分泌物中并抑制Hap自身蛋白水解， 导致表面缔合Hap的积累。在目前的建议中，我们将重点放在Hap介导的 粘附和小菌落形成。在目标1中，我们将解决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。在目标2中，我们将检查 抵抗阳离子肽的杀伤，逃避巨噬细胞的吞噬作用， 在灰鼠中耳炎模型中的持久性。在目标3中，我们将描述 呼吸道病毒感染和Hap介导的粘附和小菌落形成，集中在作用 的SLPI。 从实践的角度来看，拟议的研究可能有助于制定新的战略， H.流行性感冒也许更重要的是，他们可以提供一般 深入了解宿主-微生物关系，扩大我们对微生物生物膜的理解。

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 (HapB). Ultimately, Hap undergoes autoproteolytic cleavage, with extracellular release of Haps. In recent wor k, we demonstrated that Hap- mediated adherence and microcolony formation are potentiated by a host protein called secretory leukocyte protease 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 for the 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.