Thioredoxin mediated Acinetobacter baumannii colonization in the GI tract

 描述(由申请人提供)：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有关。这种病原体在环境中普遍存在，对干燥和抗生素具有高度抵抗力，导致许多医疗机构对入院患者采取严格的筛查方案 控制扩散。尽管大量文献表明其具有在人体胃肠道定植的能力，并且至少有一项研究提出了胃肠道定植与获得抗生素耐药性之间的联系，但尚未有关于胃肠道定植机制的实证研究报道。最近，我们的实验室着手解决这一缺陷，利用一只小鼠 口腔GI挑战赛模特。为此，我们观察到在SIgA存在的情况下，胃肠道定植和感染增加，与之前的报告相反，该细菌通过降解SIgA来中和其保护屏障功能。我们观察到免疫球蛋白的二硫键减少和分泌成分(SC)的释放。缺乏SIgA可降低IgA缺陷动物的细菌附着率和死亡率。体外实验表明，细菌酶硫氧还蛋白-A(TrxA)的基因表达上调是对SIGA暴露的响应。因此，用不对称二硫化合物(PX-12)抑制该酶，据报道可以特异性地抑制硫氧还蛋白和硫氧还蛋白折叠基序(-C-X-X-C-)，导致细菌对WT小鼠肠道上皮切片的附着减少。此外，鲍曼不动杆菌临床分离株(∆trxA)trxA基因的缺失显著降低了毒力。基于这些观察结果，我们推测鲍曼不动杆菌分泌的TrxA能降低SIgA，从而增强细菌的定植能力，因此，免疫和药物靶向TrxA为控制这种重要的MDR病原体提供了一种新的途径。这项拟议的研究将首先通过比较WT MDR临床分离株和我们的∆trxA突变体来描述TrxA介导的SC与SIgA的解离和细菌附着的增强这一新的不动杆菌致病机制来验证这一中心假设。其独特的致病机制将通过抗体抑制TrxA功能和小分子药物治疗来进一步评估/证实。拟议的抗体和药物研究的结果将为开发治疗这种重要的MDR病原体的方法(例如，单抗、改进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.