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Papers by Dominique Evans

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens and are the leadin... more Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens and are the leading causes of implant associated infections. After insertion into the body, implants become coated in host proteins, which S. aureus and S. epidermidis use to establish infections. They utilise many surface and secreted proteins that interact with these host proteins to increase attachment to implant surfaces, increase biofilm accumulation, and evade the immune system. S. aureus secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of a fibrin network that is a key structure in S. aureus biofilms and shields bacteria from the immune system. We explored which factors cause coagulase expression, the localisation and dynamics of fibrin formation in growing biofilms, and cell-cell variation in fibrin binding using coagulation assays, time lapse confocal microscopy, and single molecule imaging of Coa:SNAP, Coa:msfGFP, and vWbp:CLIP fusion proteins. Host factors increased coagulase production and loosely associated Coa and vWbp to cell surfaces. Coa mainly localised to cell surfaces to produce a surface attached fibrin pseudocapsule, but could also form fibrin in the wider biofilm matrix. vWbp produced matrix-associated fibrin in the absence of Coa, but associated to cell surfaces to accelerate pseudocapsule production when Coa was also present. These findings indicate a more collaborative role between Coa and vWbp in building the fibrin network than previously suggested. Not all bacteria appeared to contribute to forming fibrin: we identified a slowly-or non-dividing subpopulation of bacteria that did not form a pseudocapsule. We speculated that these bacteria either lack the surface proteins required to bind fibrin, do not produce coagulases and therefore cannot produce fibrin, or both. Extracellular matrix binding protein (Embp) is a giant surface protein expressed by S. epidermidis that attaches biofilms to fibronectin coated surfaces. We aimed to visualise Embp in S. epidermidis biofilms to further investigate its biological role in biofilm formation by constructing fusion proteins with the SNAP tag and monomeric superfolder GFP (msfGFP), but the fusion proteins could not be visualised. This was likely due to improper placement of the protein tags, which were placed before a putative cleavage site after the signal peptide. However, we demonstrated that msfGFP could be successfully secreted by S. aureus, either when fused to a Sec signal peptide or to Coa, which demonstrates that it is a good candidate for labelling extracellular proteins. We also visualised the SNAP tag when secreted at the cross wall during cell division by S. aureus, the same mechanism used to secrete Embp, and therefore envision that it is possible to visualise Embp with one of these two protein tags if the tag is placed after the cleavage site instead.

Microbial Cell

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secrete... more 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 t...

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens and are the leadin... more Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens and are the leading causes of implant associated infections. After insertion into the body, implants become coated in host proteins, which S. aureus and S. epidermidis use to establish infections. They utilise many surface and secreted proteins that interact with these host proteins to increase attachment to implant surfaces, increase biofilm accumulation, and evade the immune system. S. aureus secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of a fibrin network that is a key structure in S. aureus biofilms and shields bacteria from the immune system. We explored which factors cause coagulase expression, the localisation and dynamics of fibrin formation in growing biofilms, and cell-cell variation in fibrin binding using coagulation assays, time lapse confocal microscopy, and single molecule imaging of Coa:SNAP, Coa:msfGFP, and vWbp:CLIP fusion proteins. Host factors increased coagulase production and loosely associated Coa and vWbp to cell surfaces. Coa mainly localised to cell surfaces to produce a surface attached fibrin pseudocapsule, but could also form fibrin in the wider biofilm matrix. vWbp produced matrix-associated fibrin in the absence of Coa, but associated to cell surfaces to accelerate pseudocapsule production when Coa was also present. These findings indicate a more collaborative role between Coa and vWbp in building the fibrin network than previously suggested. Not all bacteria appeared to contribute to forming fibrin: we identified a slowly-or non-dividing subpopulation of bacteria that did not form a pseudocapsule. We speculated that these bacteria either lack the surface proteins required to bind fibrin, do not produce coagulases and therefore cannot produce fibrin, or both. Extracellular matrix binding protein (Embp) is a giant surface protein expressed by S. epidermidis that attaches biofilms to fibronectin coated surfaces. We aimed to visualise Embp in S. epidermidis biofilms to further investigate its biological role in biofilm formation by constructing fusion proteins with the SNAP tag and monomeric superfolder GFP (msfGFP), but the fusion proteins could not be visualised. This was likely due to improper placement of the protein tags, which were placed before a putative cleavage site after the signal peptide. However, we demonstrated that msfGFP could be successfully secreted by S. aureus, either when fused to a Sec signal peptide or to Coa, which demonstrates that it is a good candidate for labelling extracellular proteins. We also visualised the SNAP tag when secreted at the cross wall during cell division by S. aureus, the same mechanism used to secrete Embp, and therefore envision that it is possible to visualise Embp with one of these two protein tags if the tag is placed after the cleavage site instead.

Microbial Cell

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secrete... more 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 t...