Abstract
Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.
Highlights
(8) covalently attached to the peptidoglycan through the action of sortases, mainly the housekeeping transpeptidase sortase A (SrtA) [9, 10]
To enable the large-scale profiling of S. aureus adhesion to host ligands, here we describe the development of a whole cell high-throughput assay (Fig. 1A)
We selected the predominant community-associated methicillin-resistant S. aureus (MRSA) strain in North America: MRSA USA300 [59, 60], which is designated CMRSA-10 in Canada
Summary
(8) covalently attached to the peptidoglycan through the action of sortases, mainly the housekeeping transpeptidase sortase A (SrtA) [9, 10]. Existing adhesion detection methods have largely relied on staining of adhered bacteria with crystal violet, or the manual enumeration of colony-forming units [16,17,18,19,20] These assays are capable of detecting strains with reduced adhesion, they lack the feasibility, consistency, sensitivity, and/or speed required for high-throughput applications. An important component of bacterial virulence is adhesion to the host’s skin and mucosae [1,2,3,4]; as such, the development of anti-adhesive therapeutics could provide significant clinical benefits [1, 3, 5] Such an approach requires an indepth knowledge of adhesion-associated factors in clinically relevant strains [6]. The interaction of S. aureus with fibronectin underlies the organism’s ability to invade non-professional phagocytic host cells, which is mediated by the fibronectin-binding proteins (FnBPs) A and B
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