Presentations

Abstract

Introduction: Staphylococcus aureus (SA) is a major health threat due to its ability to cause disease, resist antibiotics and evade host immunity, thus highlighting the need for novel anti-staphylococcal therapies. SA virulence factors facilitate immune evasion, with many targeting the complement system, a component of innate immunity central to controlling bacterial infections. Virulence factors associated with the bacterial surface, due to their location, are ideal therapeutic targets. Previously, we demonstrated that the SA surface protein serine-aspartate repeat-protein E (SdrE) binds the complement regulator Factor H (FH) to subvert host immunity. Thus, the aim of the present study was to further explore the value of SdrE as a potential therapeutic target by creating a sdrE knockout model of S. aureus in a clinical isolate and designing a small peptide to compete with FH binding to SA SdrE.

Methods: A representative sdrE(+) community associated USA300 SA clinical isolate was selected for this study. Chromosomal deletion of sdrE (sdrE -/-) was performed using allelic exchange methodology. Briefly, an Escherichia coli-SA shuttle vector containing homologous regions to the select isolate sandwiched around a tetracycline resistance cassette (TetR) was generated via cloning methods. Plasmid elements were confirmed via Sanger sequencing, analytical digests and PCR. The generated plasmid was transformed into restriction-deficient SA RN4220, followed by bacteriophage transduction of the clinical isolate. Homologous recombination was encouraged using environmental temperature shifts, assessed by patch plating, and allelic replacement was confirmed by whole-genome sequencing (WGS). For peptide studies, two peptides were designed in silico to disrupt/compete with SdrE:FH using published interaction-domain data, applying random and targeted mutagenesis (Peptide 1) or amino-acid probability (Peptide 2) methodologies. Peptides were biotinylated for visualization purposes. Peptide-binding assays were performed using the sdrE(+) and its isogenic sdrE knockout. To further explore binding, we tested two additional clinical isolates: one that carries bbp (bone sialoprotein-binding protein), an sdrE allelic variant, and one lacking sdrE and bbp. Briefly, bound peptide was extracted from SA using 2% SDS and heat, then examined for peptide presence via bio-dot/slot-blot, probing with streptavidin-IR680. Binding was calculated using Peptide standard curves, subtracting no-treatment control samples as background.

Results: The sdrE knock-out was successfully generated, with tetR in place of sdrE. Virulence-factor genes associated with the immune evasion cluster (IEC-1) were not displaced by the genetic manipulation, as determined by WGS. Surprisingly, all isolates bound both peptides, with a dose-response curve evident. Interestingly, Peptide2 revealed a higher affinity for isolates lacking sdrE compared to those carrying sdrE or bbp.

Conclusion: Overall, these data suggest that both peptides bind to SA targets in addition to SdrE, which may be due to homologous domains present in SA proteins of the same family. Future studies will examine the effect of peptide on SA FH recruitment and complement evasion. Examining the contribution of SdrE to S. aureus pathogenesis via creation of a sdrE -/- clinical isolate model and design of peptides to disrupt the immune-evasive SdrE:FH interaction supports the advancement of better targeted anti-staphylococcal approaches.