Study of SarA by DNA Affinity Capture Assay (DACA) Employing Three Promoters of Key Virulence and Resistance Genes in Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most well-known human pathogens, houses many virulence factors and regulatory proteins that confer resistance to diverse antibiotics. Although they have been investigated intensively, the correlations among virulence factors, regulatory proteins and antibiotic resistance are still elusive. We aimed to identify the most significant global MRSA regulator by concurrently analyzing protein-binding and several promoters under same conditions and at the same time point. DNA affinity capture assay (DACA) was performed with the promoters of mecA, sarA, and sarR, all of which significantly impact survival of MRSA. Here, we show that SarA protein binds to all three promoters. Consistent with the previous reports, ΔsarA mutant exhibited weakened antibiotic resistance to oxacillin and reduced biofilm formation. Additionally, production and activity of many virulence factors such as phenol-soluble modulins (PSM), α-hemolysin, motility, staphyloxanthin, and other related proteins were decreased. Comparing the sequence of SarA with that of clinical strains of various lineages showed that all sequences were highly conserved, in contrast to that observed for AgrA, another major regulator of virulence and resistance in MRSA. We have demonstrated that SarA regulates antibiotic resistance and the expression of various virulence factors. Our results warrant that SarA could be a leading target for developing therapeutic agents against MRSA infections.

Keywords: DACA; MRSA; clinical strain; sarA; virulence factors.

Figure 1

Schematic figure of DNA affinity capture assay (DACA) and identification through Nano-liquid chromatography mass spectrometry (nLC-MS/MS) of various proteins that bind to target pro-moters (A). In DACA, each promoter region of mecA, sarA, and sarR genes was amplified using PCR before use. (B) Identification of possible regulators on each target promoter through nLC-MS/MS.

Figure 2

Antibiotic resistance of ΔsarA and changes in the expression of various virulence factors. The cell growth and biofilm formation of the JE2 and ΔsarA strains were measured in varying oxacillin concentrations. Optical densities of cells were compared at 595 nm (A). After removing the supernatant of the culture medium, the degrees of biofilm formation were compared at O.D of 595 nm through methanol fixation and crystal violet staining (B). JE2 and ΔsarA were presented in black and white circles and statistical analysis was performed by applying ANOVA with the level of significance set at 5% (A,B). Hla of JE2 and ΔsarA strains were quantified and compared by western blot immunoassay. Hla signal was hardly detected in ΔsarA unlike in JE2 (C). Each type of PSM was quantified and compared by LC-MS analysis. All types of PSM production were significantly reduced in ΔsarA (D).

Figure 3

Motility and staphyloxanthin alteration of ΔsarA. Motility tests were performed by culturing for 10 h on soft agar TSB plates. The tests were performed in triplicate with similar results (A). Cell pellets of samples harvested at each time were extracted through methanol, and the extracted staphyloxnathin were compared by measuring absorbance at 470 nm (B). JE2 and ΔsarA were presented in black and white circles and statistical analysis was performed by applying 240 ANOVA with the level of significance at 5% (B).

Figure 4

Comparison of proteomic changes between S. aureus JE2 and ΔsarA mutant. (A) Volcano plot of proteomic changes for JE2 and ΔsarA mutant. Red color indicates relatively high expression in ΔsarA mutant and blue color indicates relatively low expression in ΔsarA mutant. Proteins with fold change > 1.5 and with adjusted p-value < 0.05 were considered statistically significant (n = 4). (B) Principal component analysis (PCA) plot of proteomic analysis for JE2 and ΔsarA mutant. (C) Changes in the expression of proteins related to the virulence factor of S. aureus. The red bar indicates relatively high expression, and the blue bar indicates relatively low expression in the ΔsarA mutant compared with that of the wild type. The asterisk marks (*) indicate adjusted p-value < 0.05.

Figure 5

Alignment of SarA proteins in clinical samples.