Autosomal dominant OTULIN deficiency and staphylococcal disease

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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.