Staphylococcus aureus induced itch and neuro-immune signaling in skin infections

项目总结 瘙痒是一种令人不快的感觉，会引起抓挠的欲望。虽然瘙痒伴随着许多皮肤感染，但 微生物在瘙痒中的致病作用以前还没有被研究过。瘙痒引起的抓挠可能会导致 导致严重的皮肤损伤和细菌致病。在这里，我们研究了人类细菌的作用 致病菌金黄色葡萄球菌及其分泌的蛋白酶在驱使瘙痒和炎症中的作用。金黄色葡萄球菌 定植90%由特应性皮炎引起的皮肤损伤，特应性皮炎是一种以慢性瘙痒为特征的疾病。金黄色葡萄球菌 还会引起脓疱病，这是一种以皮肤瘙痒为特征的传染性皮肤病。我们假设金黄色葡萄球菌 分泌可直接作用于宿主感觉神经元的蛋白酶，以驱动瘙痒、抓挠引起的皮肤损伤，以及 神经免疫相声。我们的初步数据显示，小鼠皮肤表面感染金黄色葡萄球菌可诱导 强烈的瘙痒行为(同种异体膝关节、自发性瘙痒)和由此导致的皮肤病理。使用同基因突变菌株， 我们发现分泌型金黄色葡萄球菌的蛋白酶，特别是丝氨酸蛋白酶V8(SSPA)是瘙痒所必需的。 在感染期间产生。在特定的目标1中，我们将确定金黄色葡萄球菌V8蛋白酶在驱使瘙痒中的作用， 神经元激活和皮肤发炎。我们将利用同基因金黄色葡萄球菌突变株和互补株。 对于V8，以及重组V8，以阐明该蛋白酶在诱导瘙痒中的必要性和充分性， 抓伤引起损伤，金黄色葡萄球菌引起炎症。瘙痒由背根神经节(DRG)介导 感觉神经元。将进行DRG神经元钙成像以研究特定的神经元反应 V8蛋白酶。脸颊皮内注射和神经行为分析将被用来区分瘙痒和。 小鼠的疼痛行为。在特定的目标2中，我们将确定神经元和宿主细胞是否检测到金黄色葡萄球菌 V8蛋白通过特定的蛋白水解酶激活受体(PARs)。初步数据表明，PAR1可能是 一种重要的宿主受体，由V8激活。我们将利用生化和发光技术 确定PAR1上V8裂解位点的方法。我们将用药物拮抗剂治疗小鼠 抗PAR1，并利用PAR1-/-小鼠确定对V8-蛋白水解酶和金黄色葡萄球菌引起的瘙痒的影响。在……里面 具体目标3，我们将利用遗传方法来消融特定的皮肤神经细胞(Nav1.8+，MRGprd+， 和TRPV1+)，以研究它们在金黄色葡萄球菌诱导的炎症中的作用。我们假设神经元将驱动这两个 瘙痒/抓挠引起的损伤，以及直接向免疫细胞发出信号的神经介质的释放。我们会 使用有针对性的方法和蛋白质组学来评估促炎症介质的神经元释放。我们的工作 可以阐明瘙痒时金黄色葡萄球菌、宿主神经元和免疫细胞之间的新的分子串扰。三位一体 这项研究的目的是利用赵博士和霍斯威尔博士的互补技能，结合神经生物学， 免疫学和微生物学方法研究瘙痒和神经免疫的机制 金黄色葡萄球菌感染的信号。鉴于瘙痒在皮肤病中的重要性，阐明微生物在 诱导这种感觉可能会改变我们对皮肤屏障上宿主与微生物相互作用的理解。

PROJECT SUMMARY Itch is an unpleasant sensation that evokes a desire to scratch. While itch accompanies many skin infections, a causative role for microbes in itch has not been previously investigated. Itch-induced scratching can contribute to significant skin damage and bacterial pathogenesis. Here, we investigate the role of the human bacterial pathogen Staphylococcus aureus and its secreted proteases in driving itch and inflammation. S. aureus colonizes 90% of skin lesions caused by Atopic Dermatitis, a disease characterized by chronic itch. S. aureus also causes impetigo, a contagious skin disease characterized by itchy lesions. We hypothesize that S. aureus secretes proteases that can directly act on host sensory neurons to drive itch, scratch-induced skin damage, and neuroimmune crosstalk. Our preliminary data shows that S. aureus epicutaneous infection of mice induces robust itch behaviors (alloknesis, spontaneous itch) and resulting skin pathology. Using isogenic mutant strains, we find that secreted S. aureus proteases, and in particular the serine protease V8 (SspA) is required for itch production during infection. In Specific Aim 1, we will determine the role of S. aureus V8 protease in driving itch, neuronal activation, and skin inflammation. We will utilize isogenic S. aureus mutant and complemented strains for V8, as well as recombinant V8 to elucidate the necessity and sufficiency of this protease in inducing itch, scratch induced damage, and inflammation caused by S. aureus. Itch is mediated by dorsal root ganglia (DRG) sensory neurons. DRG neuron calcium imaging will be performed to investigate specific neuronal responses to V8 protease. Cheek intradermal injections and neurobehavioral analysis will be performed to distinguish itch vs. pain behaviors in mice. In Specific Aim 2, we will determine whether neurons and host cells detect S. aureus V8 protease through specific protease-activated receptors (PARs). Preliminary data indicates that PAR1 may be an important host receptor that is activated by V8. We will utilize biochemical and luminescence-based approaches to determine the V8 cleavage site on PAR1. We will treat mice with pharmacological antagonists against PAR1 and utilize PAR1-/- mice to determine effects on V8-protease and S. aureus induced itch. In Specific Aim 3, we will utilize genetic approaches to ablate specific skin-innervating neurons (Nav1.8+, Mrgprd+, and Trpv1+) to assay their roles in S. aureus induced inflammation. We hypothesize that neurons will drive both itch/scratch-induced damage, and the release of neural mediators that directly signal to immune cells. We will use targeted approaches and proteomics to assess neuronal release of proinflammatory mediators. Our work could elucidate novel molecular crosstalk between S. aureus, host neurons and immune cells in itch. The three aims of this study leverage the complementary skills of Dr. Chiu and Dr. Horswill, combining neurobiological, immunological, and microbiological approaches to investigate the mechanisms of itch and neuroimmune signaling in S. aureus infection. Given the importance of itch in skin diseases, elucidating a microbial role in inducing this sensation could transform our understanding of host-microbe interactions at the skin barrier.