Abstract
The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.
Highlights
Staphylococcus aureus is a major nosocomial and community acquired pathogen causing a diverse array of infections ranging from superficial infections of the skin and mucosa to highly invasive and potentially lethal infections
RNA-Seq showed that the saeSP allele of strain Newman affects expression of nearly 125 genes, relative to strains with the saeSL allele
The RNA-Seq analyses confirmed that the saeSP allele has a profound effect on gene expression, including numerous genes that may play a role in biofilm formation
Summary
Staphylococcus aureus is a major nosocomial and community acquired pathogen causing a diverse array of infections ranging from superficial infections of the skin and mucosa to highly invasive and potentially lethal infections. Many bacterial infections are associated with biofilm formation. Formation of biofilms occurs in distinct phases of attachment, accumulation and disassembly [1,4,5,6,7]. The attachment phase is often mediated by bacterial cell surface proteins, collectively known as MSCRAMMs, which bind to host plasma/extracellular matrix proteins such as fibronectin, fibrinogen and collagen [8]. The accumulation step in biofilm formation requires mechanisms for intercellular aggregation. PNAG-containing biofilms often contain bacterial proteins and extracellular DNA. The composition of staphylococcal biofilms is quite variable, with some strains being heavily dependent on PNAG and other strains being more dependent on extracellular proteins and DNA [4,6,9]
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