Autosomal dominant OTULIN deficiency and staphylococcal disease

项目摘要 金黄色葡萄球菌是影响人类健康的主要病原菌。然而，尽管大多数受试者携带S。 金黄色葡萄球菌，只有少数人会患上危及生命的葡萄球菌疾病。我们假设严重的或复发的 葡萄球菌感染，至少在一些患者中，可能是由单基因先天性免疫缺陷（IEI）引起的。 我们已经发现NF-κ B和TLR 2相关基因的IEIs易患葡萄球菌感染。作为 本项目的一个基础是，一组100名儿童和年轻人患有严重或复发性S。金黄色葡萄球菌感染 已经使用全外显子组测序（WES）进行了遗传分析，该技术已在实验室中率先用于 IEIs。在全基因组范围内寻找候选基因型，通过测试各种遗传模型，我们 在来自6个激酶的7个患者中发现了OTULIN的杂合罕见非同义变体。OTULIN，a 已知去泛素酶调节炎症，但不支配抗葡萄球菌免疫。双等位 OTULIN中的有害变体引起称为OTULIN相关的自身炎症的严重早发性病症 奥拉斯综合征。有趣的是，OTULIN位于5号染色体的短臂上， 5 p-（Cri-du-Chat）综合征，一种患病率为1/30，000的遗传性疾病。5 p综合征患者 容易受到细菌感染。我们假设常染色体显性OTULIN缺乏症通过以下方式起作用： 单倍不足和葡萄球菌疾病的基础。初步结果显示，这6名患者的等位基因是 4例杂合子患者皮肤成纤维细胞中OTULIN功能丧失和表达 当来自1名患有5 β-综合征（具有缺失的OTULIN拷贝）的患者的OTULIN变体时， 与健康对照组相比。此外，在真皮中观察到线性泛素链的积累， 来自3名患者的成纤维细胞，包括一名5 β-综合征患者。最后，我们注意到了一个小窝蛋白的积累， 已知其促进ADAM 10的募集，ADAM 10是主要葡萄球菌毒力因子a的受体， 溶血素因此，我们观察到患者的成纤维细胞对a-溶血素具有高度的选择性敏感性 细胞毒因此，本申请的目的是更深入地探索OTULIN的具体作用 在来自OTULIN有害变体杂合子患者的成纤维细胞和单核细胞中。我们将调查 OTULIN对真皮成纤维细胞中线性泛素组的影响，特别强调NF-κ B和 使用转录组学和蛋白质组学方法检测TLR 2下游的炎症标志物。我们还将 研究患者白细胞亚群的天然免疫应答。最后，我们将研究内在的 在小窝蛋白失调的情况下，患者对细菌毒素（包括α-溶血素）的免疫力， ADAM 10表达。总的来说，这项研究将重视新兴的概念，即细菌感染可以 在遗传性免疫缺陷的情况下优先引发严重疾病。这个项目将揭示 关于葡萄球菌疾病的发病机制和OTULIN的生物学，同时为新型预防性药物铺平道路。 以及用于OTULIN突变的患者，包括患有5 β-综合征的患者的治疗措施。

PROJECT SUMMARY Staphylococcus aureus is a major bacterial pathogen impacting human health. Yet, while most subjects carry S. aureus, only a minority develop life-threatening staphylococcal disease. We hypothesize that severe or recurrent staphylococcal infections, at least in some patients, can result from single-gene inborn errors of immunity (IEI). We have already shown that IEIs in NF-kB- and TLR2-related genes predispose to staphylococcal infections. As a basis for this project, a cohort of 100 children and young adults with severe or recurrent S. aureus infections has been analyzed genetically using whole exome sequencing (WES), which has been pioneered in the lab for IEIs. Searching for candidate genotypes in a genome-wide manner, by testing various genetic models, we discovered heterozygous rare non-synonymous variants in OTULIN in 7 patients from 6 kindreds. OTULIN, a deubiquitinase, is known to regulate inflammation but not to govern anti-staphylococcal immunity. Bi-allelic deleterious variants in OTULIN cause a severe early-onset condition termed OTULIN-related autoinflammatory syndrome (ORAS). Interestingly, OTULIN is located on the short arm of the chromosome 5 and is often deleted in the 5p- (Cri-du-Chat) Syndrome, a genetic disease with a prevalence of 1/30,000. Patients with 5p- Syndrome are susceptible to bacterial infections. We hypothesize that autosomal dominant OTULIN deficiency operates by haploinsufficiency and underlies staphylococcal disease. Preliminary results show that the 6 patients’ alleles are loss-of-function and that expression of OTULIN in dermal fibroblasts from 4 patients carrying heterozygous OTULIN variants and from 1 patient with 5p- Syndrome (with a missing copy of OTULIN) is about half, when compared with healthy controls. Moreover, accumulation of linear ubiquitin chains was observed in dermal fibroblasts from 3 patients, including one with the 5p- Syndrome. Finally, we noticed an accumulation of Caveolin, which is known to facilitate recruitment of ADAM10, the receptor for the major staphylococcal virulence factor a- hemolysin. Consistently, we observed a high and selective susceptibility of patients’ fibroblasts to a-hemolysin cytotoxicity. The aim of the present application is therefore to explore in more depth the specific role of OTULIN in fibroblasts and monocytes from patients heterozygous for deleterious variants in OTULIN. We will investigate the impact of OTULIN on the linear ubiquitinome in dermal fibroblasts, with a special emphasis on NF-kB and markers of inflammation downstream TLR2 using transcriptomic and proteomics approaches. We will also investigate the innate immune responses in patients’ leukocyte subsets. Finally, we will investigate the intrinsic immunity of patients to bacterial toxins, including a-hemolysin, in the context of Caveolin dysregulation and ADAM10 expression. Overall, this study will give weight to the emerging notion that bacterial infection can preferentially trigger severe disease in the context of genetically deficient immunity. This project will shed light on the pathogenesis of staphylococcal disease and biology of OTULIN, while paving the way for novel preventive and therapeutic measures for OTULIN-mutated patients, including those with the 5p- Syndrome.