pubmed.ncbi.nlm.nih.gov

Commensal Staphylococcus epidermidis contributes to skin barrier homeostasis by generating protective ceramides - PubMed

  • ️Sat Jan 01 2022

Commensal Staphylococcus epidermidis contributes to skin barrier homeostasis by generating protective ceramides

Yue Zheng et al. Cell Host Microbe. 2022.

Abstract

Previously either regarded as insignificant or feared as potential sources of infection, the bacteria living on our skin are increasingly recognized for their role in benefitting human health. Skin commensals modulate mucosal immune defenses and directly interfere with pathogens; however, their contribution to the skin's physical integrity is less understood. Here, we show that the abundant skin commensal Staphylococcus epidermidis contributes to skin barrier integrity. S. epidermidis secretes a sphingomyelinase that acquires essential nutrients for the bacteria and assists the host in producing ceramides, the main constituent of the epithelial barrier that averts skin dehydration and aging. In mouse models, S. epidermidis significantly increases skin ceramide levels and prevents water loss of damaged skin in a fashion entirely dependent on its sphingomyelinase. Our findings reveal a symbiotic mechanism that demonstrates an important role of the skin microbiota in the maintenance of the skin's protective barrier.

Keywords: Staphylococcus epidermidis; ceramides; commensal; probiotic; skin; skin barrier; skin microbiota; symbiosis.

Copyright © 2022. Published by Elsevier Inc.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.. S. epidermidis skin colonization decreases water loss and increases stratum corneum ceramide content.

A, Epidermal architecture, role of aSMase, and proposed role of the S. epidermidis sphingomyelinase Sph in homeostasis of the skin protective barrier. The graph shows the different layers of the epidermis, with live keratinocytes in the lower and dead corneocytes in the outmost stratum corneum layer. The acid sphingomyelinase (aSMase) enzyme, which produces phosphocholine (PC) and ceramide (Cer) from sphingomyelin is produced in keratinocytes and secreted via exocytosis in lamellar bodies. As described in the present study, S. epidermidis colonizes the skin surface and secretes an enzyme, Sph, with the same activity, assisting aSMase in the production of the skin’s protective barrier that is composed of lipid lamellae of ceramides, cholesterol, and free fatty acids (FA). The SM and Cer type shown in the insert is the most abundant Cer 2 type (d18:1/16:0), with the hydroxyl group shown in red present in the Cer 5 type. d, 1,3-dihydroxy. B, Setup of mouse model. A/E, acetone/diethyl ether. SDS, sodium dodecyl sulfate. TEWL, transepidermal water loss. TEWL measurements 24 h after skin compromise and association of mice with the indicated bacteria or PBS as control. Treatment groups were formed by evenly distributing mice according to their initial TEWL levels due to high mouse-specific variations in TEWL background levels. n=14/group. C, Total stratum corneum ceramide amount after association of compromised skin of live mice for 48 h with S. epidermidis or PBS control (n=8/group). C,D, Statistical analysis is by unpaired t-tests. Error bars show the mean ± SD.

Fig. 2.
Fig. 2.. Enzymatic activity, species-wide and in-vivo expression of Sph.

A, Cleavage of sphingomyelin from human keratinocytes by purified Sph (100 μg/ml). Shown is the Tandem-MS analysis of the major sphingomyelin type with d18:1 sphingosine, producing type-2 ceramide. FA, fatty acid; Sp, sphingosine (d18:1) of undetermined FA chain length; dh, dihydro (d18:0 instead of d18:1); P, phosphate. n=3. See Fig. S2 for results with other tissue sources using RP-HPLC/ESI-MS. B, In-vitro expression of Sph by Western blot and relative densitometric analysis in a collection of S. epidermidis isolates. Filled colored circles show data obtained with strains 1457 (red) and S25 (orange), open circles those with strains 1457 Δsph (red) and S25 Δsph (orange). See Fig. S4 for Western blot images. C, Production of ceramides by culture filtrates of a collection of S. epidermidis isolates. Filled colored circles show data obtained with strains 1457 (red) and S25 (orange), open circles those with strains 1457 Δsph (red) and S25 Δsph (orange). D, SM degradation and Cer production by other skin-colonizing bacteria compared to S. epidermidis. n=3. Statistical analysis is by 1-way ANOVA with Dunnett’s post-test via data obtained with TSB. E, Expression of the sph gene on mouse skin associated with the S. epidermidis strains used in this study. Expression was determined 1 day after association. n=6/group. F, Expression of the sph gene in swabs obtained from armpits and faces of human volunteers. n=20. Computation of mean and SD includes 0 values from individuals without detectable expression (ND). A-F, Error bars show the mean ± SD. S. epid., S. epidermidis; Cut. acnes, Cutibacterium acnes; Cor. amycolatum, Corynebacterium amycolatum.

Fig. 3.
Fig. 3.. S. epidermidis Sph does not harm the host.

A, Lysis of keratinocytes by the indicated bacterial strains. Bacteria were seeded at a ratio of 2:1 to keratinocytes. After 1-day incubation, the final ratio of bacteria to keratinocytes was about 700:1. Cytotoxicity is expressed as percent relative to complete lysis with triton X100 (100% lysis). n=3/group. B, Lysis of human erythrocytes by the indicated bacteria as measured by heme release. The bacteria to erythrocyte ratio was 5000:1. n=8/group. See Fig. S5 for results with sheep erythrocytes and bacterial culture filtrates. C, Biofilm formation in TSB/0.125% glucose. Biofilms were grown for 24 h in microtiter plates, stained with crystal violet, and absorption at 560 nm was measured. n=8/group. See Fig. S5 for results with different glucose concentrations. D, Comparison of enzymatic activities (sphingomyelin cleavage) of purified Sph and S. aureus β-toxin at equal concentrations (1.5 μg/ml). n=3/group. E, Binding of S. epidermidis Sph and S. aureus β-toxin to liposomes and keratinocytes. Binding was determined and measured via Western Blots and densitometry of positive bands. n=6/group. Statistical analysis is by two-tailed, unpaired t-tests. F, Structure of S. epidermidis Sph modeled using SWISS MODEL (

https://swissmodel.expasy.org

) based on the closest related sequence available (PDB 2uyr.1.A; B. cereus sphingomyelinase N57A). The three non-conserved amino acids of the edge metal binding site are shown with side chains (K91, V132, S133; see Fig. S1). The solvent-exposed loop is highlighted in yellow. Illustrations of the model obtained by SWISS MODEL were performed using Protean 3D (Lasergene 17). G, Structure comparison of S. epidermidis Sph with B. cereus Sph (PDB 2uyr.1.A) and S. aureus β-toxin (PDB 3i5v.1.A). Blue color designates conserved, red color divergent parts. A-E, Error bars show the mean ± SD. S. e., S. epid., S. epidermidis; S. c., S. carnosus.

Fig. 4.
Fig. 4.. Sph promotes S. epidermidis growth under skin-like conditions and during in-vivo skin colonization.

A, Growth in synthetic medium (SNLM) under low-nutrient conditions of S. epidermidis 1457 wild-type (WT) and isogenic sph mutant (Δsph), with or without sphingomyelin (SM) (n=3/group). B, Effect of genetic complementation under the same conditions (with SM; n=3/group). C, Growth of Δsph mutant under the same conditions (without SM) with and without addition of PC; n=6/group. D, Growth of WT and Δsph mutant in SNLM at physiological (125 mM, n=3/group) and high (2 M, n=6/group) concentrations of NaCl with or without SM. E, Growth of genetic complementation and corresponding control strains in SNLM with 2 M NaCl (n=5/group) with or without SM. F, Growth of Δsph mutant in SNLM with 2 M NaCl with and without addition of PC (without SM). n=8/group. G,H, CFU measurements 1 day (24 h) or 5 days (120 h) after association of mice with equal CFU (2 × 104/cm2) of WT or isogenic Δsph bacteria of strains S. epidermidis 1457 (n=8/group, 1 day; n=16/group, 5 days) or S25 (n=8/group, 1 day; n=4–6/group, 5 days). I, Complementation of 1457 Δsph strain in-vivo growth deficiency with PC. 50 μg of PC was applied to the skin of mice. (n=6/group, 1 day; n=16/group, 5 days). B,E, Plasmid-harboring strains were grown with addition of tetracycline (12.5 μg/ml). C,D,F,G,H, I, Statistical analysis is by unpaired, two-tailed t-tests. A,B,E, Statistical analysis is by 1-way ANOVAs with Tukey’s post-tests. A-I, Error bars show the mean ± SD. S. epid., S. epidermidis.

Fig. 5.
Fig. 5.. S. epidermidis produces ceramides via Sph from host cells and on the skin in vivo.

A, SM degradation and B, ceramide production [measuring the main SMase-derived ceramide type 2 (d18:1/16:0, see Figs. 1,2)] in human keratinocyte culture by equal CFU (107) of S. epidermidis wild-type (WT), sph isogenic mutant (Δsph), sph-complemented and control strains in S. epidermidis 1457 and S25 strain backgrounds. n=4/group (1457); n=3/group (S25). C, Setup of mouse experiment used for the results shown in panels D-F. A/E, acetone/diethyl ether. Mouse picture is from

Stockio.com

. D, Stratum corneum ceramide (d18:1/16:0) production by purified Sph on the compromised skin of live mice. n=8/group. E, Stratum corneum ceramide 2 (d18:1/16:0) production, after association with compromised skin of live mice for 24 or 48 h (n=7), respectively, with the indicated bacteria or PBS as control. F, Total stratum corneum ceramide production after association of compromised mouse skin for 48 h with the indicated bacteria (n=8/group; measurement by TLC). G, Expression of host ceramide biosynthesis genes. n=3/group. D, Statistical analysis is by unpaired, two-tailed t test. A,B,E,F,G, statistical analysis is by one-way ANOVAs with Tukey’s post-tests. A,B,D,E,F,G, Error bars show the mean ± SD.

Fig. 6.
Fig. 6.. S. epidermidis promotes skin rehydration after compromise via Sph production in vivo.

A, Setup of mouse model used for the results shown in panels B-E. A/E, acetone/diethyl ether. B, Transepidermal water loss (TEWL) measurements 24 h after application of Sph. n=8/group. C, TEWL 24 h after association of mice with the indicated bacteria or PBS as control. n=14/group (1457); n=6/group (S25). D, Effect of treatment of compromised skin of live mice with ceramide 2 or PBS as control, TEWL measurements. n=6/group. E, Effect of treatment of compromised skin of live mice previously associated with S. epidermidis 1457 Δsph with ceramide 2, TEWL measurements. n=6/group. F, Atopic dermatitis (AD) model used for the results shown in panels G and H. Mice were treated to develop inflammatory phenotypes typical for AD before application of bacteria and TEWL was measured 24 h afterwards. G, IgE levels on day 22 before application of bacteria. n=5 (control group), n=3 (treatment group). See Fig. S6 and Table S1 for histological evaluation of the AD model. H, TEWL 24 h after association of mice with the indicated bacteria or PBS as control. n=10/group A-E, H, Treatment groups were formed by evenly distributing mice according to their initial TEWL levels due to high mouse-specific variations in TEWL background levels. Statistical analysis is by unpaired t-tests (A,C,D,G) or 1-way ANOVAs with Tukey post-tests (B,E,H). Error bars show the mean ± SD.

Comment in

Similar articles

Cited by

References

    1. Ago H, Oda M, Takahashi M, Tsuge H, Ochi S, Katunuma N, Miyano M, and Sakurai J (2006). Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus. J Biol Chem 281, 16157–16167. 10.1074/jbc.M601089200. - DOI - PubMed
    1. Allgaier H, Jung G, Werner RG, Schneider U, and Zahner H (1986). Epidermin: sequencing of a heterodetic tetracyclic 21-peptide amide antibiotic. Eur J Biochem 160, 9–22. 10.1111/j.1432-1033.1986.tb09933.x. - DOI - PubMed
    1. Augustin J, and Gotz F (1990). Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol Lett 54, 203–207. 10.1016/0378-1097(90)90283-v. - DOI - PubMed
    1. Bae T, and Schneewind O (2006). Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 55, 58–63. 10.1016/j.plasmid.2005.05.005. - DOI - PubMed
    1. Becker K, Heilmann C, and Peters G (2014). Coagulase-negative staphylococci. Clin Microbiol Rev 27, 870–926. 10.1128/CMR.00109-13. - DOI - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources