Staphylococcus aureus Keratinocyte Invasion Is Dependent upon Multiple High-Affinity Fibronectin-Binding Repeats within FnBPA

Andrew M Edwards,Nicola A G Meenan,Ruth C Massey,Ursula Potter,Jennifer R Potts,Malcolm James Horsburgh

doi:10.1371/journal.pone.0018899

Andrew M Edwards, Nicola A G Meenan + Show 4 more

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https://doi.org/10.1371/journal.pone.0018899

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Journal: PLoS ONE	Publication Date: Apr 22, 2011
Citations: 115	License type: CC BY 4.0

Affiliation: University of Bath, University of York

Abstract

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α5β1 integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.

Highlights

Staphylococcus aureus is a bacterium responsible for a wide range of superficial and invasive infections ranging in severity from mild to fatal [1]
This study demonstrated that a single high-affinity fibronectin-binding repeats (FnBRs) was sufficient to trigger invasion, this was less efficient than FnBPA variants containing multiple FnBRs [17]
Recombinant FnBR expression A polypeptide corresponding to high-affinity repeats FnBR9,10 (FnBPA residues 763–838; Swiss-Prot entry P14738) was expressed and purified in a manner similar to that previously described for Species/strain/plasmid E. coli K12 ER2925 BL21 pGEX-6P-2 Staphylococcus aureus DU5883 DU5883

Summary

Introduction

Staphylococcus aureus is a bacterium responsible for a wide range of superficial and invasive infections ranging in severity from mild to fatal [1]. Originally considered an extracellular pathogen, there is both in vitro and in vivo evidence that S. aureus invades host cells. The role of invasion in colonisation and infection is unclear, it is hypothesised to facilitate evasion of immune surveillance, traversal of cellular barriers, evasion of antimicrobial therapy and to enable persistent infection [9,10,11,12,13,14,15,16,17]. There is evidence that S. aureus is able to dramatically alter its phenotype (to the small colony variant phenotype) to enhance survival within host cells, which is associated with persistent infections [14,15]

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