Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus

Abstract

We have previously shown that lysates of Lacticaseibacillus rhamnosus GG confer protection to human keratinocytes against Staphylococcus aureus. L. rhamnosus GG inhibits the growth of S. aureus as well as competitively excludes and displaces the pathogen from keratinocytes. In this study, we have specifically investigated the anti-adhesive action. We have tested the hypothesis that this activity is due to quenching of S. aureus binding sites on keratinocytes by molecules within the Lacticaseibacillus lysate. Trypsinisation or heat treatment removed the protective effect of the lysate suggesting the involvement of proteins as effector molecules. Column separation of the lysate and analysis of discrete fractions in adhesion assays identified a fraction of moderate hydrophobicity that possessed all anti-adhesive functions. Immunoblotting demonstrated that this fraction contained the pilus protein, SpaC. Recombinant SpaC inhibited staphylococcal adhesion to keratinocytes in a dose-dependent manner and improved keratinocyte viability following challenge with viable S. aureus. However, SpaC did not confer the full anti-adhesive effects of the LGG lysate and excluded but did not displace S. aureus from keratinocytes. Further purification produced four protein-containing peaks (F1–F4). Of these, F4, which had the greatest column retention time, was the most efficacious in anti-staphylococcal adhesion and keratinocyte viability assays. Identification of proteins by mass spectrometry showed F4 to contain several known “moonlighting proteins”—i.e., with additional activities to the canonical function, including enolase, Triosephosphate isomerase (TPI), Glyceraldehyde 3 phosphate dehydrogenase (G3P) and Elongation factor TU (EF-Tu). Of these, only enolase and TPI inhibited S. aureus adhesion and protected keratinocytes viability in a dose-dependent manner. These data suggest that inhibition of staphylococcal binding by the L. rhamnosus GG lysate is mediated by SpaC and specific moonlight proteins.

Postbiotic lactobacilli induce cutaneous antimicrobial response and restore the barrier to inhibit the intracellular invasion of Staphylococcus aureus in vitro and ex vivo

“…The antimicrobial effect of the BGMK2‐41 postbiotic was demonstrated on two strains of intracellular S. aureus , chronic wound isolate DUM2 and USA300‐GFP. In contrast to a previously reported mechanism of inhibition of pathogen adhesion to keratinocytes by Lacticaseibacillus rhamnosus GG lysate, 20 we were able to demonstrate induction of AMPs post‐treatment, targeting and clearing already internalized S. aureus . In addition to a reduction of intracellular S. aureus load in vitro, we also confirmed antimicrobial activity of the BGMK2‐41 postbiotic against extracellular S. aureus in a human wound model ex vivo, suggesting the therapeutic potential against both externally colonizing and intracellular residing S. aureus .…”

“…Topical treatment of keratinocytes in vitro with lactobacilli lysates has shown anti‐inflammatory properties, 13 stimulation of migration, bolstering the epithelial barrier, and protection against S. aureus adhesion 14–18 . Lacticaseibacillus rhamnosus GG lysate can accelerate wound closure in vitro via CXCL2/CXCR2 and prevents initial binding of S. aureus to keratinocytes 17,19,20 . However, the therapeutic effects of postbiotics generated from lactobacilli to effectively clear residing intracellular S. aureus as seen in cutaneous infections have not been tested before and is the focus of this study.…”

Lactobacillus rhamnosus GG triggers intestinal epithelium injury in zebrafish revealing host dependent beneficial effects

“…In this study, the oral administration of SpaC at a level of approximately 5 or 10 μg per day per zebrafish induced intestinal mucosa damage and pyroptosis in zebrafish. SpaC is intolerant to trypsinisation treatment and its degradation products may not necessarily possess the same activity or efficacy as the intact SpaC protein [ 46 ]. Thus, we can conclude that the degradation products of SpaC did not participate in inducing intestinal mucosa damage and pyroptosis in zebrafish.…”

Molecular Mechanisms of Lacticaseibacillus rhamnosus, LGG® Probiotic Function

“…SpaC was involved in this function, as recombinant SpaC inhibited S. aureus adhesion to keratinocytes in a dose-dependent manner and improved keratinocyte viability following S. aureus challenge. However, enolase and triosephosphate isomerase present in the LGG ® lysate were likely also involved in these effects [114].…”