Research Career Development: Investigation of novel Staphylococcus aureus alpha-toxin targets at the cellular adherens junctions

 描述（由申请方提供）：金黄色葡萄球菌既是一种常见的皮肤定植菌，也是一种强大的病原体，在美国引起大多数皮肤和软组织以及手术部位感染。S.金黄色葡萄球菌皮肤感染作为一个网站， 细菌几乎可以传播到任何器官。这些侵入性感染每年在美国造成约11，000人死亡。此外，抗生素耐药菌株（MRSA）治疗起来特别困难和昂贵，并构成不断升级的公共卫生威胁。了解S。金黄色葡萄球菌持续存在并破坏皮肤上皮，有可能提供巨大的预防和治疗价值。 我们对这一重要病原体的认识存在重大差距，金黄色葡萄球菌毒力因子改变了宿主上皮生态位，使其长期组织定植，以及S.金黄色葡萄球菌在从定植到感染的转变过程中破坏上皮屏障。分泌的成孔毒力因子A-毒素靶向细胞-细胞连接蛋白E-钙粘蛋白进行切割，并且是S.金黄色葡萄球菌致病过程中上皮感染。在上皮细胞-细胞连接处阻断α-毒素活性可能最终是干扰MRSA皮肤定殖和随后感染到更深组织中的有效策略。 考虑到这一点，我们进行了一个新的宿主因子所需的a-毒素毒性的致醉诱变的单倍体人类细胞的遗传筛选。我们的筛选鉴定了一组编码上皮粘附连接成员的基因，这些基因在缺失时赋予对α-毒素的抗性。我们已经验证了我们的屏幕上发现的最突出的粘附连接基因的作用，促进α-毒素的细胞毒性。我们假设，改变该基因的表达将降低a毒素在皮肤中的毒力，并消除MRSA感染的发病率。在这个建议中，我们将使用体外人类细胞系，三维人类表皮器官型皮肤培养模型，和转基因小鼠研究的后果，a-毒素毒力介导的细胞粘附连接。该F31培训奖将提供必要的实验资源，时间和培训支持，以确定粘附连接如何控制MRSA致病性。这些实验的结果可能会导致更好的策略来预防和治疗MRSA感染，这是国家疾病控制的优先事项。

 DESCRIPTION (provided by applicant): Staphylococcus aureus is both a common skin colonizer and also a formidable pathogen, causing the majority of skin and soft tissue and surgical site infections in the United States. S. aureus skin infections serve as a site from which bacteria can disseminate to virtually any organ. These invasive infections cause an estimated 11,000 deaths in the U.S. annually. In addition, antibiotic- resistant strains (MRSA) are especially difficult and costly to treat and constitute an escalating public health threat. Understanding how S. aureus persists in and breaches the skin epithelium has the potential to deliver tremendous preventative and therapeutic value. Significant gaps in our knowledge of this important pathogen are understanding how S. aureus virulence factors modify the host epithelial niche for long-term tissue colonization and how S. aureus breaches epithelial barriers during the transition from colonization to infection. The secreted pore- forming virulence factor a-toxin targets the cell-cell junction protein E-cadherin for cleavage and is essential for S. aureus pathogenesis during epithelial infections. Blocking a-toxin activity at the epithelial cell-cell junctions may ultimately be an effective strategy to interfere with MRSA skin colonization and subsequent infection into deeper tissues. With this idea in mind, we performed a genetic screen for novel host factors required for a-toxin virulence by intoxicating mutagenized haploid human cells. Our screen identified a set of genes that encode for members of the epithelial adherens junctions which, when absent, confer resistance to a- toxin. We have validated the most prominent adherens junction gene discovered by our screen for its role in facilitating a-toxin cytotoxicity. We hypothesize that altering expression of this gene will reduce a-toxin virulence in the skin and abrogate morbidity from MRSA infection. In this proposal, we will use in vitro human cell lines, a 3D human epidermal organotypic skin culture model, and a transgenic mouse to study the consequences of a-toxin virulence mediated by the cellular adherens junctions. This F31 training award will provide the experimental resources, time, and training support necessary to identify how the adherens junctions control MRSA pathogenicity. Results from these experiments will likely lead to better strategies to prevent and treat MRSA infections, a national priority for disease control.