Expression of sap Operon in NTHI-induced Otitis Media

描述(由申请人提供)：细菌已经进化出抵抗宿主防御机制的策略，包括对抗菌肽(AP)的抗药性。SAP操纵子(对抗菌肽的敏感性)操纵子在这种防御中发挥着重要作用，动物模型中SAP操纵子突变体的衰减证明了这一点。这一设想的假设是，SAP操纵子通过在体内维持对AP的抗性和上调特定AP耐药决定因素的表达，在非分型流感嗜血杆菌(NTHI)中耳炎模型中发挥重要作用。用流式细胞术和组织学方法分析SAP操纵子在中耳炎病程中的表达，以及在体外暴露于栗鼠中性粒细胞或中性粒细胞颗粒提取物时的表达。NTHI汁液操纵子的六个基因的突变将定义体内每个基因的功能需求。最后，微阵列分析将被用来确定由于汁液操纵子表达而决定对AP的抗性的基因。这些研究产生的数据将极大地促进该领域理解NTHI如何适应疾病进展过程中遇到的微环境中的体内先天免疫反应，并将识别由汁液操纵子表达导致的新的毒力机制，使NTHI能够存活和致病。

DESCRIPTION (provided by applicant): Bacteria have evolved strategies to resist host defense mechanisms including resistance to antimicrobial peptides (APs). The sap (sensitivity to antimicrobial peptides) operon plays a significant role in this defense, as demonstrated by attenuation of sap operon mutants in animal models. The hypothesis of this proposal is that the sap operon plays a significant role in nontypeable Haemophilus influenzae (NTHI) survival in a chinchilla model of otitis media by maintaining resistance to APs in vivo and by up-regulating expression of specific AP resistance determinants. Expression of the sap operon during the disease course of otitis media or when exposed in vitro to chinchilla neutrophils or neutrophil granule extract will be analyzed by flow cytometry and histology. Mutations in the six genes of the NTHI sap operon will define the functional requirement for each gene in vivo. Finally, microarray analysis will be used to identify genes that determine resistance to APs as a result of sap operon expression. The data generated from these studies will significantly advance the field in understanding how NTHI adapt to the innate immune response in vivo in microenvironments encountered during disease progression and will identify novel virulence mechanisms resulting from sap operon expression that enable NTHI to survive and cause disease.