GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms
Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they mus...
| Auteurs principaux: | , , , , , , , , , |
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| Format: | Article |
| Langue: | English |
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Shared Science Publishers OG
2023-06-01
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| Collection: | Microbial Cell |
| Sujets: | |
| Accès en ligne: | http://microbialcell.com/researcharticles/2023a-evans-microbial-cell/ |
| _version_ | 1826532034085912576 |
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| author | Dominique C. S. Evans Amanda B. Khamas Lisbeth Marcussen Kristian S. Rasmussen Janne K. Klitgaard Birgitte H. Kallipolitis Janni Nielsen Daniel E. Otzen Mark C. Leake Rikke L. Meyer |
| author_facet | Dominique C. S. Evans Amanda B. Khamas Lisbeth Marcussen Kristian S. Rasmussen Janne K. Klitgaard Birgitte H. Kallipolitis Janni Nielsen Daniel E. Otzen Mark C. Leake Rikke L. Meyer |
| author_sort | Dominique C. S. Evans |
| collection | DOAJ |
| description | Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they must be ex-ported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detect-ed msfGFP fluorescence inside but not outside bacterial cells, indicating a fail-ure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically inte-grated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus. |
| first_indexed | 2024-03-13T02:55:26Z |
| format | Article |
| id | doaj.art-51cdcdeead1b4d71959554d9bbf38cf9 |
| institution | Directory Open Access Journal |
| issn | 2311-2638 |
| language | English |
| last_indexed | 2025-03-14T01:44:48Z |
| publishDate | 2023-06-01 |
| publisher | Shared Science Publishers OG |
| record_format | Article |
| series | Microbial Cell |
| spelling | doaj.art-51cdcdeead1b4d71959554d9bbf38cf92025-03-12T13:46:42ZengShared Science Publishers OGMicrobial Cell2311-26382023-06-0110714515610.15698/mic2023.07.800GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilmsDominique C. S. Evans0Amanda B. Khamas1Lisbeth Marcussen2Kristian S. Rasmussen3Janne K. Klitgaard4Birgitte H. Kallipolitis5Janni Nielsen6Daniel E. Otzen7Mark C. Leake8Rikke L. Meyer9Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.Department of Physics, University of York, York, UK.Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they must be ex-ported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detect-ed msfGFP fluorescence inside but not outside bacterial cells, indicating a fail-ure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically inte-grated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.http://microbialcell.com/researcharticles/2023a-evans-microbial-cell/fusion proteingram positive bacteriamonomeric superfolder gfpcoagulasebiofilms |
| spellingShingle | Dominique C. S. Evans Amanda B. Khamas Lisbeth Marcussen Kristian S. Rasmussen Janne K. Klitgaard Birgitte H. Kallipolitis Janni Nielsen Daniel E. Otzen Mark C. Leake Rikke L. Meyer GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms Microbial Cell fusion protein gram positive bacteria monomeric superfolder gfp coagulase biofilms |
| title | GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms |
| title_full | GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms |
| title_fullStr | GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms |
| title_full_unstemmed | GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms |
| title_short | GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms |
| title_sort | gfp fusions of sec routed extracellular proteins in staphylococcus aureus reveal surface associated coagulase in biofilms |
| topic | fusion protein gram positive bacteria monomeric superfolder gfp coagulase biofilms |
| url | http://microbialcell.com/researcharticles/2023a-evans-microbial-cell/ |
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