Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus
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 sp...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.875542/full |
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| author | Cecile El-Chami Rawshan Choudhury Walaa Mohammedsaeed Andrew J. McBain Veera Kainulainen Sarah Lebeer Reetta Satokari Catherine A. O’Neill |
| author_facet | Cecile El-Chami Rawshan Choudhury Walaa Mohammedsaeed Andrew J. McBain Veera Kainulainen Sarah Lebeer Reetta Satokari Catherine A. O’Neill |
| author_sort | Cecile El-Chami |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T09:23:19Z |
| format | Article |
| id | doaj.art-6359c965a1764b12a1c3c3e6ba7e500c |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-13T09:23:19Z |
| publishDate | 2022-05-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-6359c965a1764b12a1c3c3e6ba7e500c2022-12-22T02:52:32ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-05-011310.3389/fmicb.2022.875542875542Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureusCecile El-Chami0Rawshan Choudhury1Walaa Mohammedsaeed2Andrew J. McBain3Veera Kainulainen4Sarah Lebeer5Reetta Satokari6Catherine A. O’Neill7Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, United KingdomFaculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, United KingdomFaculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, United KingdomFaculty of Biology, School of Health Sciences, Medicine and Health, University of Manchester, Manchester, United KingdomFaculty of Medicine, Human Microbiome Research Program, University of Helsinki, Helsinki, FinlandDepartment of Bioscience Engineering, University of Antwerp, Antwerp, BelgiumFaculty of Medicine, Human Microbiome Research Program, University of Helsinki, Helsinki, FinlandFaculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, United KingdomWe 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.https://www.frontiersin.org/articles/10.3389/fmicb.2022.875542/fullprobiotickeratinocyteLacticaseibacillus rhamnosus GGSpaCmoonlight proteinsStaphylococcus aureus |
| spellingShingle | Cecile El-Chami Rawshan Choudhury Walaa Mohammedsaeed Andrew J. McBain Veera Kainulainen Sarah Lebeer Reetta Satokari Catherine A. O’Neill Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus Frontiers in Microbiology probiotic keratinocyte Lacticaseibacillus rhamnosus GG SpaC moonlight proteins Staphylococcus aureus |
| title | Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus |
| title_full | Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus |
| title_fullStr | Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus |
| title_full_unstemmed | Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus |
| title_short | Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus |
| title_sort | multiple proteins of lacticaseibacillus rhamnosus gg are involved in the protection of keratinocytes from the toxic effects of staphylococcus aureus |
| topic | probiotic keratinocyte Lacticaseibacillus rhamnosus GG SpaC moonlight proteins Staphylococcus aureus |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.875542/full |
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