Role of OxyR in P. aeruginosa Biofilm Resistance to H202

描述(申请人提供)：在囊性纤维化(CF)呼吸道疾病中，有令人信服的证据表明存在两个不同的临床阶段，即氧化期(早期CF)和厌氧期(慢性晚期CF)。在CF呼吸道内厚厚的粘液中，细菌以“生物膜”的形式生长，这种发展形式为生物体提供了增强对抗生素和杀菌剂的抵抗力的奢侈。早期的氧化阶段是基于中性粒细胞迅速和戏剧性地进入上呼吸道，这是由细菌感染引发的事件。当受到刺激时，这些专业吞噬细胞会产生一种强大的“呼吸爆发”，一种来自过氧化氢(H202)的抗菌产物。事实上，中性粒细胞可以在吞噬液泡中产生毫米级的H202。相比之下，血液中的H202水平几乎低1000倍。令人惊讶的是，主要CF病原体铜绿假单胞菌的突变株缺乏H202反应的反式激活剂OxyR，它对H202非常敏感，即使在血液中H202水平(微摩尔范围)存在的情况下，细菌也会死亡。如果OxyR在人类感染期间受到损害，细菌将无法引发系统性感染，因为它们将通过H202介导的杀戮而死亡。因此，本提案的目的是确定铜绿假单胞菌的OxyR是否可以作为各种铜绿假单胞菌感染的药物靶点。这项建议的目标是(I)定义OxyR在动物毒力和对人类中性粒细胞耐药中的作用；(Ii)定义在OxyR突变体中引起对H202高度敏感的病变以及哪些OxyR控制的基因产品有助于最大或最低限度的保护；以及(Iii)确定OxyR是否对生物被膜细菌对H202和氨基糖苷类药物的生存至关重要。

DESCRIPTION (provided by applicant): In cystic fibrosis (CF) airway disease, there is compelling evidence for two distinct clinical stages, an oxidative phase (early-stage CF) and an anaerobic phase (chronic, late-stage CF). Within the thick mucus lining the CF airways, the bacteria grow as a "biofilm," a form of development that affords organisms the luxury of enhanced resistance to antibiotics and biocides. The early oxidative phase is based upon a rapid and dramatic influx of neutrophils to the upper airways, an event triggered by bacterial infection. When stimulated, these professional phagocytes mount a potent "respiratory burst," an antimicrobial product from which is hydrogen peroxide (H202). In fact, neutrophils can generate millimolar levels of H202 within the phagolysomal vacuole. In contrast, H202 levels in blood are nearly 1000-fold lower. Surprisingly, a mutant of the major CF pathogen, Pseudomonas aeruginosa, lacking the H202-responsive transactivator, OxyR, is exquisitely sensitive to H202 and the bacteria perish even in the presence of blood H202 levels (micromolar range). Were OxyR to be compromised during human infection, bacteria would be unable to elicit a systemic infection because they would die via H202-mediated killing. Therefore, the goal of this proposal is to determine if OxyR of P. aeruginosa could serve as a drug target during various P. aeruginosa infections. The goals of this proposal are to (i) define a role for OxyR in animal virulence and resistance to human neutrophils, (ii) define the lesions that evoke exquisite sensitivity to H202 in the OxyR mutant and what OxyR-controlled gene products contribute to maximal or minimal protection, and (iii) determine whether OxyR is critical for survival of biofilm bacteria to H202 and aminoglycosides.