Molecular mechanisms of staphylococcal modulation of skin homeostasis - PROJECT SUMMARY / ABSTRACT Staphylococcus (S.) sp. are ubiquitous colonizers of human skin and mucosa. Its commensal members, such as S. epidermidis, make key contributions to skin health by modulating the immune system and promoting skin microbiome homeostasis. Other species, such as S. aureus, can be asymptomatic colonizers or disease- causing pathobionts. While S. hominis, capitis, haemolyticus and other species are ubiquitous, they are little characterized vis a vis their role in the skin microbiome. Yet in our extensive surveys of healthy and diseased skin, we have found significant shifts in the species level composition of skin staphylococci. For example, healthy skin strongly favors colonization with epidermidis and hominis, while patients with congenital ichthyosis shifts dramatically to a capitis-dominated microbiome. Eczema associated with primary immunodeficiencies is also more prominently characterized by non-aureus staphylococci, including haemolyticus. Finally, in atopic dermatitis, strain-level differences in epidermidis can be associated with active disease, not only the canonical aureus. These species and strain-level differences are striking in their diversity, and we hypothesize that genetically diverse Staphylococcus species and strains differentially affect skin homeostasis by altering the inflammatory milieu and/or skin barrier. The goal of this proposal is to investigate how diverse staphylococcal species and strains drive epidermal responses. We amassed and sequenced a collection of thousands of staphylococcal isolates from human skin, and examined the transcriptional response of 3D human reconstructed skin equivalents (RHE) to colonization with 180 of these diverse species and strains, identifying species- and strain-level differences in the skin's response. Here, we will build on this preliminary data to investigate host and microbial mechanisms underlying these interactions. Selecting a subset of 30 isolates that produced signatures of interest from our RHE screen, we will examine transcriptional response in a fibroblast+RHE (F-RHE) model from multiple matched donors to dissect how increased skin complexity modifies the colonization response (Aim 1). We will use RNA-seq, scRNA-seq, spatial transcriptomics, and proteomics to isolate cell-type and layer-specific responses to colonization, as well as reconstruct likely cell-cell signaling pathways. Second, given the recently identified impact of commensal microbiota on skin barrier and immunity mediated through AhR, we will similarly investigate the ability of these staphylococci to modulate the skin barrier by producing metabolites that activate AhR (Aim 2). Using our established metabolomics and CRISPRi toolkits developed in staphylococci, we will investigate microbial mechanisms to identify likely involved metabolites. These experiments will further our goal to investigate how diverse staphylococcal species and strains drive epidermal responses, and will allow us to define mechanisms of a microbial contribution to numerous inflammatory skin diseases that affect the skin barrier.
