GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureusreveal surface-associated coagulase in biofilms

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*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review


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 chal-lenges of using fluorescent protein reporters in their native environment, be-cause they must be exported 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 ma-jor 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 detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicat-ing successful export of the msfGFP in the unfolded state, followed by extra-cellular 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 bacte-ria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the bio-film matrix. Our findings demonstrate that msfGFP is a good candidate fluo-rescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

Original languageEnglish
JournalMicrobial Cell
Pages (from-to)145-156
Number of pages12
Publication statusPublished - Jul 2023


  • biofilms
  • coagulase
  • fusion protein
  • Gram positive bacteria
  • monomeric superfolder GFP


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