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

Massey RC (2011) Staphylococcus aureus Keratinocyte Invasion Is Dependent upon Multiple High-Affinity Fibronectin-Binding Repeats within FnBPA. PLoS ONE 6(4): e18899. doi:10.1371/journal.pone.0018899 Staphylococcus aureus Keratinocyte Invasion Is Dependent upon Multiple High-Affinity Fibronectin- Binding Repeats within FnBPA Andrew M. Edwards 0 Ursula Potter 0 Nicola A. G. Meenan 0 Jennifer R. Potts 0 Ruth C. Massey 0 Malcolm James Horsburgh, University of Liverpool, United Kingdom 0 1 Department of Biology and Biochemistry, University of Bath , Bath , United Kingdom , 2 Microscopic Analysis Suite , University of Bath , Bath , United Kingdom , 3 Department of Biology, University of York, York, United Kingdom, 4 Department of Chemistry, University of York , York , United Kingdom 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 a5b1 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 highaffinity 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. - Funding: JRP acknowledges the British Heart Foundation (http://www.bhf.org.uk/) for a Senior Basic Science Research Fellowship. This work was funded by the Wellcome Trust (http://www.wellcome.ac.uk/), grant number WT 079588. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Staphylococcus aureus is a bacterium responsible for a wide range of superficial and invasive infections ranging in severity from mild to fatal [1]. In addition to causing severe morbidity and mortality in the healthcare environment, S. aureus is a growing problem in the community, causing serious infections in otherwise healthy people [2,3]. Treatment of S. aureus infections is often complicated by the high prevalence of antibiotic resistant strains [4,5]. Despite the ability of this organism to cause serious illness, S. aureus is primarily a commensal organism, residing within the nares and on the skin of 2060% of the population either permanently or transiently [6]. Colonisation of the skin can lead to a number of persistent or recurring infections including, folliculitis, scalded skin syndrome, impetigo, colonisation of indwelling medical devices and wound infections [1,7,8]. Although originally considered an extracellular pathogen, there is both in vitro and in vivo evidence that S. aureus invades host cells. Although 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 [917]. Indeed, 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]. The primary mechanism by which S. aureus enters host cells is well characterised; staphylococcal fibronectin binding proteins (FnBPs) interact with cell surface a5b1 integrins via a fibronectin bridge [1820]. It appears that FnBPA alone is sufficient for invasion since heterologous expression on the surface of otherwise non-invasive Lactococcus lactis or Staphylococcus carnosus confers the ability to invade host cells [19]. The indirect interaction of FnBPA with a5b1 integrins leads to cell signalling events, actin rearrangement and internalization of the bacterium via a mechanism that is entirely dependent on host-cell processes [11,18,21]. FnBPs are multifunctional proteins, comprised of distinct regions with variable binding activity. The N-terminal domain binds both fibrinogen and elastin and is implicated in biofilm formation [22,23]. This region is followed by 11 (FnBPA) or 10 (FnBPB) non-identical fibronectin-binding repeats (FnBRs), with either high or low-affinity for fibronectin [24]. These multiple repeats enable a single FnBPA molecule to bind multiple fibronectin molecules [2527]. It has been hypothesised that this facilitates bacterial interactions with multiple integrins, triggering cell signalling processes, actin rearrangement and bacterial internalisation [2729]. We have previously shown that this region is essential for triggering bacterial invasion of endothelial cells [17,30]. In addition to its role in adhesion, invasion and biofilm formation, the high prevalence of fnb genes amongst S. aureus strains suggest that FnBPs might be important for colonisation; analysis of a panel of 163 clinical isolates revealed that 22% encoded just fnbA, 1% just fnbB and 77% encoded both genes [31]. We recently investigated how the composition of the FnBR region of FnBPA affected the invasion of endothelial cells and virulence in a murine bacteremia model [17]. This study demonstrated that a single high-affinity FnBR was sufficient to trigger invasion, although this was less efficient than FnBPA variants containing multiple FnBRs [17]. Multiple FnBRs were required for virulence, enhancing bacterial dissemination into the kidneys, as well as leading to significant weight loss and death [17]. Although it is important that we understand how this protein contributes to invasive infection, this (...truncated)