Drugs from bugs: YopM - a bacteria-derived anti-inflammatory cell-penetrating peptide for the topical treatment of psoriasis

在小肠结肠炎耶尔森氏菌感染期间，细菌通过III型分泌系统将不同的耶尔森氏菌外蛋白（Yops）转运到宿主细胞质中。与其他Yops相比，YopM不具有任何酶活性，并且已证明在转运到宿主细胞后，YopM通过囊泡相关途径转运到细胞核。在细胞核中，YopM抑制编码促炎细胞因子的基因的转录，如tnf - α、IL-1 β、ifn - γ、IL-12或IL-18。因此，在鼠疫杆菌感染的小鼠模型中，YopM降低了巨噬细胞中这些促炎细胞因子的表达，导致细菌的毒力增加，并降低了宿主的抗菌免疫。然而，到目前为止，尚不清楚局部易位效应蛋白如何对抗菌免疫反应的全身效应负责。有趣的是，在之前的研究中，我们发现YopM是一种细胞穿透肽，并证明其能够自主进入各种哺乳动物细胞，包括免疫细胞和上皮细胞，通过小窝蛋白依赖的内吞作用，然后是内体逃逸，而无需其他因素的帮助。为了支持这一点，用重组YopM （rYopM）治疗活化的原代免疫细胞导致tnf - α， il -1 β和ifn - γ的下调，这表明rYopM作为一种自我传递的抗炎药物，因此可能对炎症性疾病的治疗感兴趣。为了研究rYopM在体内的抗炎和治疗潜力，我们采用吡喹莫德诱导的银屑病小鼠模型。通常，免疫调节剂，皮质类固醇或生物制剂用于治疗牛皮癣。然而，这些药物中的许多在局部应用时无效，因为它们无法穿透表皮屏障。由于rYopM突破屏障进入宿主细胞而不需要其他因素，我们使用含有rYopM的乳膏局部治疗银屑病小鼠，令人惊讶的是，可以显示表皮涂抹rYopM显着减少银屑病的所有特征，如棘皮病或乳头状瘤病。现在，我们打算分析局部rYopM治疗小鼠和人类皮肤改善银屑病的细胞和分子机制。在组织培养模型的帮助下，我们将分析rYopM对信号网络的影响，包括皮肤屏障功能以及先天和适应性免疫反应。此外，我们将通过定点诱变产生截断的rYopM蛋白，以确定穿透上皮屏障和抗炎活性所需的结构域。因此，将确定具有改进的免疫调节能力和增加的稳定性的rYopM变体，这可能为局部免疫治疗的新型治疗替代方案的发展奠定基础。

During Yersinia enterocolitica infection bacteria translocate different Yersinia outer proteins (Yops) into the host cell cytoplasm through a type III secretion system. YopM, in contrast to other Yops, does not possess any enzymatic activity and it has been shown that after translocation into the host cell, YopM traffics to the nucleus via a vesicle-associated pathway. In the nucleus YopM inhibits the transcription of genes encoding pro-inflammatory cytokines such as TNF-alpha, IL 1beta, IFN-gamma, IL-12, or IL-18. Accordingly, in a mouse model of Y. pestis infection YopM reduced the expression of these pro-inflammatory cytokines in macrophages leading to an increased virulence of bacteria as well as a down-regulated anti-bacterial immunity in the host. However, thus far it is not known how a locally translocated effector protein might be responsible for systemic effects on anti-bacterial immune responses. Interestingly, in previous studies we identified YopM as a cell-penetrating peptide and demonstrated its ability to autonomously enter various mammalian cells including immune and epithelial cells via caveolin-dependent endocytosis followed by endosomal escape and without the help of additional factors. In support of this, treatment of activated primary immune cells with recombinant YopM (rYopM) led to the down-regulation of TNF-alpha, IL-1beta and IFN-gamma indicating that rYopM acts as a self-delivering anti-inflammatory drug and therefore, might be of interest for the treatment of inflammatory disorders. To investigate the anti-inflammatory and therapeutic potential of rYopM in vivo we used the mouse model of imiquimod-induced psoriasis. Usually, immunomodulators, corticosteroids or biologics are used for the treatment of psoriasis. However, many of these drugs are ineffective when applied locally because of their inability to penetrate the epidermal barrier. As rYopM breaches barriers and enters host cells without the requirement of additional factors we topically treated psoriatic mice with a rYopM-containing cream and surprisingly, could show that epicutaneous application of rYopM markedly reduced all characteristic hallmarks of psoriasis like acanthosis or papillomatosis. Now, we intend to analyze the cellular and molecular mechanisms underlying the amelioration of psoriasis by topical rYopM treatment in mouse as well as human skin. With the help of tissue culture models the effect of rYopM on signaling networks will be analyzed, including the skin barrier function and the innate as well as adaptive immune response. Furthermore, we will generate truncated rYopM proteins by site-directed mutagenesis to identify domains required for the penetration of epithelial barriers as well as the anti-inflammatory activity. Thereby, rYopM variants with improved immunomodulatory capacities and increased stability will be determined, which might represent the basis for the development of a novel therapeutic alternative for local immunotherapy.