Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

BACKGROUND & AIM: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. RESULTS: All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. CONCLUSION: Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Reduced filaggrin expression is accompanied by increased Staphylococcus aureus colonization of epidermal skin models.

BACKGROUND: Atopic dermatitis is an inflammatory skin disease that is characterized by a reduced skin barrier function, reduced filaggrin (FLG) expression as well as increased colonization by Staphylococcus aureus. OBJECTIVE: This study focused on the possible involvement of FLG in epidermal colonization by S. aureus and/or whether it affects the epidermal defence mechanisms, including the expression of antimicrobial peptides (AMPs) and enzymes involved in stratum corneum barrier lipid synthesis. Furthermore, IL-31 has been shown to reduce FLG expression, but its effects on bacterial colonization and on the expression of AMPs and enzymes involved in the barrier lipid synthesis are not known. MATERIAL AND METHODS: We established N/TERT-based epidermal models (NEMs), after FLG knockdown (FLG-KD) and/or cultured with IL-31, that were colonized with S. aureus for 24 h. RESULTS: Both FLG-KD and IL-31 supplementation resulted in significantly increased epidermal S. aureus colonization, as well as in an up-regulation of S. aureus-induced IL-8 expression. IL-31, but not FLG-KD, prevented S. aureus-induced up-regulation of mRNA expression for the AMPs human ß-defensin 2 and -3 and RNAse7, whereas psoriasin expression remained unchanged. Furthermore, the S. aureus colonization induced changes in mRNA expression of ELOVL4 was not affected by FLG-KD, but was blocked by IL-31. Expression of SCD-1 and Gcase mRNA was reduced by IL-31, but not by FLG-KD. CONCLUSION: This study shows that NEMs, with FLG-KD and/or cultured in the presence of IL-31, mimic the skin of patients with atopic dermatitis in several aspects, including enhanced bacterial colonization, increased inflammatory and reduced protective responses.

Anti-cyclic citrullinated peptide antibodies from rheumatoid arthritis patients activate complement via both the classical and alternative pathways.

OBJECTIVE: It has been suggested that anti-citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). To exert their pathologic effects, ACPAs must recruit immune effector mechanisms such as activation of the complement system. Mouse models of RA have shown that, surprisingly, arthritogenic antibodies activate the alternative pathway of complement rather than the expected classical pathway. This study was undertaken to investigate whether human anti-cyclic citrullinated peptide (anti-CCP) antibodies activate the complement system in vitro and, if so, which pathways of complement activation are used. METHODS: We set up novel assays to analyze complement activation by anti-CCP antibodies, using cyclic citrullinated peptide-coated plates, specific buffers, and normal and complement-deficient sera as a source of complement. RESULTS: Anti-CCP antibodies activated complement in a dose-dependent manner via the classical pathway of complement, and, surprisingly, via the alternative pathway of complement. The lectin pathway was not activated by anti-CCP antibodies. Complement activation proceeded in vitro up to the formation of the membrane attack complex, indicating that all activation steps, including the release of C5a, took place. CONCLUSION: Our findings indicate that anti-CCP antibodies activate the complement system in vitro via the classical and alternative pathways but not via the lectin pathway. These findings are relevant for the design of interventions aimed at inhibition of complement-mediated damage in RA.