Thioredoxin mediated Acinetobacter baumannii colonization in the GI tract

 描述（由申请方提供）：鲍曼不动杆菌是一种主要的医院和战斗相关病原体，主要与呼吸道和伤口感染相关。这种病原体在环境中无处不在，对干燥和抗生素具有高度抗性，导致许多医疗机构对入院的患者采取严格的筛查方案， 控制蔓延。尽管有大量文献证明其能够定植于人类胃肠道，并且至少有一项研究提出胃肠道定植与抗生素耐药性的获得之间存在联系，但尚未报告研究胃肠道定植机制的实证研究。最近，我们的实验室开始利用小鼠来解决这个缺点。 口服GI激发模型。为此，我们观察到在SIgA存在下增强的胃肠道定殖和感染，与先前的报告相反，该报告表明细菌蛋白水解降解SIgA以中和其保护屏障功能。我们观察到二硫键的还原和分泌成分（SC）从免疫球蛋白中的释放。SIgA缺乏导致伊加缺乏动物的细菌附着和死亡率降低。体外试验表明，细菌酶硫氧还蛋白-A（TrxA）的基因表达上调响应SIgA暴露。因此，用不对称二硫化物化合物（PX-12）抑制这种酶，据报道特异性抑制含硫氧还蛋白和硫氧还蛋白折叠基序（-C-X-X-C-）的蛋白质，导致细菌对从WT小鼠获得的肠上皮切片的附着减少。此外，从MDR A.鲍曼不动杆菌临床分离株（BcltrxA）的毒力显著降低。基于这些观察，我们假设A.鲍曼不动杆菌分泌的TrxA增强细菌定殖，因此免疫和药物靶向TrxA提供了控制这种重要MDR病原体的新方法。拟议的研究将通过首先描述TrxA介导的SC从SIgA解离和细菌附着增强的这种新型不动杆菌致病机制，使用WT MDR临床分离株与我们的TrxA突变体的比较来验证这一中心假设。通过抗体和小分子药物治疗抑制TrxA功能，将进一步评估/证实独特的致病机制。拟定抗体和药物研究的结果将为开发这种重要MDR病原体的治疗性治疗（例如单克隆抗体、具有改善效力的精制小分子不对称化合物）提供基础。

 DESCRIPTION (provided by applicant): Acinetobacter baumannii is a major nosocomial and combat related pathogen primarily associated with respiratory and wound infections. Ubiquitous in the environment, this pathogen is highly resistant to desiccation and antibiotics leading many healthcare institutions to adopt strict screening protocols of patients admitted to the hospital to control spread. Despite extensive literature demonstrating its ability to colonize the human GI tract, and at least one study proposing a link between GI tract colonization and acquisition of antibiotic resistance, no empirical studies have been reported to study the mechanism of GI tract colonization. Recently, our laboratory set out to address this shortcoming utilizing a murine oral GI challenge model. To that end, we observed enhanced GI tract colonization and infection in the presence of SIgA, contrary to previous reports suggesting the bacteria proteolytically degraded SIgA to neutralize its protective barrier function. We observed reduction of the disulfide bonds and release of secretory component (SC) from the immunoglobulin. Absence of SIgA resulted in both decreased bacterial attachment and mortality in IgA deficient animals. In vitro assays showed that gene expression of the bacterial enzyme thioredoxin-A (TrxA) was up regulated in response to SIgA exposure. Consequently, inhibition of this enzyme with an asymmetric disulfide compound (PX-12), reported to specifically inhibit thioredoxin and thioredoxin-fold motif (-C-X-X-C-) containing proteins, resulted in decreased bacterial attachment to intestinal epithelial sections obtained from WT mice. Furthermore, deletion of the trxA gene from a MDR A. baumannii clinical isolate (∆trxA) significantly reduced virulence. Based on these observations, we hypothesize that reduction of SIgA by A. baumannii secreted TrxA enhances bacterial colonization, thus immuno- and drug-targeting TrxA provides a novel approach to control of this important MDR pathogen. The proposed study will test this central hypothesis by first delineation of this novel Acinetobacter pathogenic mechanism of TrxA-mediated dissociation of SC from SIgA and enhancement of bacterial attachment, using comparisons of WT MDR clinical isolate with our ∆trxA mutant. The unique pathogenic mechanism will be further assessed/confirmed with inhibition of TrxA function by antibody and small molecule drug treatment. The results of the proposed antibody and drug studies will provide a foundation for the development of therapeutic treatments (e.g. monoclonal antibodies, refined small molecule asymmetric compounds with improved potency) for this important MDR pathogen.