Staphylococcus aureus Enters Hair Follicles Using Triacylglycerol Lipases Preserved through the Genus Staphylococcus

The bacteria belonging to the genus Staphylococcus are a group of organisms that readily inhabit human skin and the upper respiratory tract. Staphylococcus aureus (SA) is a leading cause of soft tissue infections and bacteremia (Miller and Cho, 2011Miller L.S. Cho J.S. Immunity against Staphylococcus aureus cutaneous infections.Nat Rev Immunol. 2011; 11: 505-518Crossref PubMed Scopus (270) Google Scholar) but is closely related to many other species of Staphylococcus that are skin commensals and rarely cause disease. These species densely populate follicular structures that may serve as a reservoir for long-term colonization (Nakatsuji et al., 2013Nakatsuji T. Chiang H.I. Jiang S.B. Nagarajan H. Zengler K. Gallo R.L. The microbiome extends to subepidermal compartments of normal skin.Nat Commun. 2013; 4: 1431Crossref PubMed Scopus (283) Google Scholar). Furthermore, there is growing evidence that various other microorganisms live in hair follicles (HFs) and interact with skin immune cells (Chen et al., 2018Chen Y.E. Fischbach M.A. Belkaid Y. Skin microbiota-host interactions.Nature. 2018; 553: 427-436Crossref PubMed Scopus (265) Google Scholar). For example, Cutibacterium acnes produces short-chain fatty acids by fermenting the lipids in the sebum, and these substances have an influence on host immune function (Sanford et al., 2019Sanford J.A. O'Neill A.M. Zouboulis C.C. Gallo R.L. Short-chain fatty acids from Cutibacterium acnes activate both a canonical and epigenetic inflammatory response in human sebocytes.J Immunol. 2019; 202: 1767-1776Crossref PubMed Scopus (33) Google Scholar). Thus, it is important to further understand the mechanisms by which both pathogenic and commensal microbes establish colonization of the HF. In this study, we sought to understand how Staphylococcus can enter the HF. Because sebum is secreted from sebaceous glands and fills in the infundibulum of HFs, we hypothesized that sebum functions as a hydrophobic barrier, whereas lipases may enable bacteria to enter the HF independently of the penetration of the stratum corneum. We focused on triacylglycerol lipases that degrade triglycerides, which are the main components (60%) of sebum (Picardo et al., 2009Picardo M. Ottaviani M. Camera E. Mastrofrancesco A. Sebaceous gland lipids.Dermatoendocrinol. 2009; 1: 68-71Crossref PubMed Google Scholar), and studied the response to the deletion mutants of the SA known lipases. SA produces two secreted lipases termed gehA (SAL-1) and gehB (SAL-2), and the expressions of these genes are under the control of the accessory gene regulator (agr) quorum sensing system (Horswill and Gordon, 2020Horswill A.R. Gordon C.P. Structure-activity relationship studies of small molecule modulators of the staphylococcal accessory gene regulator.J Med Chem. 2020; 63: 2705-2730Crossref PubMed Scopus (19) Google Scholar). We compared the capacity of a methicillin-resistant SA USA300 LAC wild-type (SA WT) parent strain, a double lipase mutant SA (SA Δlipases) strain, a SA agr mutant strain, and a mutant SA strain of all 10 known secreted proteases (proteases mutant SA [SA Δproteases]) to induce skin damage and inflammation. Topically applied SA WT induced inflammation, erythema, and crust within 48 hours; SA Δlipases and SA Δproteases strains induced less inflammation, and the agr mutant was the least capable of promoting inflammation (Figure 1a). Measurement of Il6 mRNA and transepidermal water loss of the skin revealed that murine Il6 mRNA in the whole skin was decreased by 56% in the SA Δlipases strain compared with that in the SA WT strain control (Figure 1b). Transepidermal water loss was decreased by 47% in the SA Δlipases strain compared with that in the SA WT strain (Figure 1c). Overall, these data reveal how SA lipases play a role in skin inflammation and damage and revealed that these likely cooperate with other previously discovered agr-regulated skin-damaging factors such as SA proteases (Kolar et al., 2013Kolar S.L. Ibarra J.A. Rivera F.E. Mootz J.M. Davenport J.E. Stevens S.M. et al.Extracellular proteases are key mediators of Staphylococcus aureus virulence via the global modulation of virulence-determinant stability.Microbiologyopen. 2013; 2: 18-34Crossref PubMed Scopus (111) Google Scholar). Next, to determine whether SA lipases allow for penetration of microbes into the HF, the SA WT and mutant strains were applied topically to shaved back skin at 1 × 106 colony-forming unit/cm2 for 2 hours. The 16S ribosomal RNA abundance was quantified in sequential 20 μm horizontal sections of frozen skin. This analysis revealed that the SA Δlipases strain as well as the SA agr mutant and SA Δproteases strains all lacked the ability to penetrate past 100 μm of the skin surface (Figure 1d). This depth of penetration corresponds to the depth of the infundibulum that is typically within approximately 100 μm of the surface. Furthermore, DNA for the SA WT and SA Δproteases strains appeared greater within the 60–100 μm depth than for the SA Δlipases and SA agr mutant strains. This suggested that SA lipases play a role in the initial penetration of bacteria into the lipid-rich infundibulum. To confirm this, immunostaining of murine skin sections was done with an antibody specific to SA, and it revealed that only SA WT and SA Δproteases strains were frequently observed to infiltrate into the infundibulum, but mutants lacking lipases or an active agr system (lacking the capacity to secrete lipases and proteases) could not (Figure 1e). Furthermore, by counting individual staining within multiple follicles and applying the agr and total protease mutant compared with specific SA mutants to either lipase gehA or lipase gehB, we quantified what fraction of total bacterial staining could be seen within the infundibulum. This analysis revealed that SAL-2was the primary SA lipase driving initial HF penetration (Figure 1f and g). Overall, these data suggest that the specific SA lipase gene, gehB, is essential for penetration into the upper HF of mice. Lipases are widely expressed throughout the genus Staphylococcus. In the case of SA, SAL-1 (encoded by gehA) primarily reacts with short-chain glycerides, whereas SAL-2 (encoded by gehB) hydrolyzes both short-chain and long-chain triglycerides (Cadieux et al., 2014Cadieux B. Vijayakumaran V. Bernards M.A. McGavin M.J. Heinrichs D.E. Role of lipase from community-associated methicillin-resistant Staphylococcus aureus strain USA300 in hydrolyzing triglycerides into growth-inhibitory free fatty acids.J Bacteriol. 2014; 196: 4044-4056Crossref PubMed Scopus (50) Google Scholar). Two equivalent lipases are reported in S. epidermidis: SEL-1 (encoded by gehC) and SEL-2 (encoded by gehD) (Longshaw et al., 2000Longshaw C.M. Farrell A.M. Wright J.D. Holland K.T. Identification of a second lipase gene, gehD, in Staphylococcus epidermidis: comparison of sequence with those of other staphylococcal lipases.Microbiology (Reading). 2000; 146: 1419-1427Crossref PubMed Scopus (56) Google Scholar). Other staphylococcal species are known to secrete lipases, and some of their genomes are sequenced. To reveal the evolutionary differences of major lipases among the genus Staphylococcus, we performed a database search and analyzed their similarity using National Center for Biotechnology Information Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov/BLAST), followed by the analysis conducted in Molecular Evolutionary Genetics Analysis X software (www.megasoftware.net). As expected, our data suggested that lipases are highly preserved among those species and that all the staphylococcal lipases are produced as pre-proenzymes with a signal peptide in the preregion and are secreted as proenzymes needing a specific cleavage for mature configuration. The amino acid positions of the catalytic triad (serine-histidine-aspartate motif) are quite similar among the mature lipases (Figure 2a). Generally, lipases are members of a large group of enzymes possessing the α and β hydrolase folds, with the preserved arrangement of the catalytic residue (histidine-serine-aspartate or cysteine) located in a tight loop after the β5 strand (Ollis et al., 1992Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. et al.The alpha/beta hydrolase fold.Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1821) Google Scholar). In addition, the calculation indicated that on the basis of the similarity, they are categorized into two groups: one including SAL-1 and the other including SAL-2 (Figure 2b), indicating that all staphylococcal species encode two lipases. This high identity of lipases in the genus Staphylococcus implies important roles in metabolism and adaptation to the environment (Nguyen et al., 2018Nguyen M.T. Luqman A. Bitschar K. Hertlein T. Dick J. Ohlsen K. et al.Staphylococcal (phospho)lipases promote biofilm formation and host cell invasion.Int J Med Microbiol. 2018; 308: 653-663Crossref PubMed Scopus (23) Google Scholar) and suggests that our observations in SA may apply to other species of Staphylococcus in the HF. These observations add important new insight into the previously known contributions of SA proteases to skin damage (Nakatsuji et al., 2016Nakatsuji T. Chen T.H. Two A.M. Chun K.A. Narala S. Geha R.S. et al.Staphylococcus aureus exploits epidermal barrier defects in atopic dermatitis to trigger cytokine expression.J Invest Dermatol. 2016; 136: 2192-2200Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar; Williams et al., 2019Williams M.R. Costa S.K. Zaramela L.S. Khalil S. Todd D.A. Winter H.L. et al.Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis.Sci Transl Med. 2019; 11eaat8329Crossref PubMed Scopus (89) Google Scholar) and suggest that the expression of lipases by Staphylococcus enables bacteria within this genus to establish residence in the HF. No datasets were generated during this study. All mouse procedures were approved by the University of California San Diego Institutional Animal Care and Use Program (Protocol Number: S09074). Kouki Nakamura: http://orcid.org/0000-0002-2296-6004 Michael R. Williams: http://orcid.org/0000-0002-1523-6353 Jakub M. Kwiecinski: http://orcid.org/0000-0001-9472-2896 Alexander R. Horswill: http://orcid.org/0000-0002-5568-0096 Richard L. Gallo: http://orcid.org/0000-0002-1401-7861 RLG is a cofounder, scientific advisor, and consultant; has equity in MatriSys Biosciences; and is a consultant, receives income from, and has equity in Sente Inc. The remaining authors state no conflict of interest. KN was supported by grants from Uehara Memorial Foundation , Japan. RLG, MRW, and ARH are supported by the National Institute of Health grant R01AI153185. MRW is supported by the National Institute of General Medical Sciences training grant 5K12GM068524-18. RLG is supported by the National Institute of Health grants R01AR074302, R01AR076082, R37AI052453, and U01AI52038. Conceptualization: KN, MRW, RLG; Formal Analysis: KN, MRW; Investigation: KN, MRW, JMK; Supervision: RLG; Writing – Original Draft Preparation: KN, MRW, RLG; Writing – Review and Editing: KN, MRW, JMK, ARH, RLG