Salicylic acid diminishes Staphylococcus aureus capsular polysaccharide type 5 expression

Abstract

Capsular polysaccharides (CP) of serotypes 5 (CP5) and 8 (CP8) are major Staphylococcus aureus virulence factors. Previous studies have shown that salicylic acid (SAL), the main aspirin metabolite, affects the expression of certain bacterial virulence factors. In the present study, we found that S. aureus strain Reynolds (CP5) cultured with SAL was internalized by MAC-T cells in larger numbers than strain Reynolds organisms not exposed to SAL. Furthermore, the internalization of the isogenic nonencapsulated Reynolds strain into MAC-T cells was not significantly affected by preexposure to SAL. Pretreatment of S. aureus strain Newman with SAL also enhanced internalization into MAC-T cells compared with that of untreated control strains. Using strain Newman organisms, we evaluated the activity of the major cap5 promoter, which was significantly decreased upon preexposure to SAL. Diminished transcription of mgrA and upregulation of the saeRS transcript, both global regulators of CP expression, were found in S. aureus cultured in the presence of SAL, as ascertained by real-time PCR analysis. In addition, CP5 production by S. aureus Newman was also decreased by treatment with SAL. Collectively, our data demonstrate that exposure of encapsulated S. aureus strains to low concentrations of SAL reduced CP production, thus unmasking surface adhesins and leading to an increased capacity of staphylococci to invade epithelial cells. The high capacity of internalization of the encapsulated S. aureus strains induced by SAL pretreatment may contribute to the persistence of bacteria in certain hosts.

FIG. 1.

Effect of SAL on internalization of S. aureus into MAC-T epithelial cells. Confluent MAC-T cells were tested for staphylococcal invasion. Each bar represents the “percentage of invasion,” defined as the median of intracellular CFU/ml (n = 9 to 12) from S. aureus strains related to the median of intracellular CFU/ml from S. aureus RN6390 or Reynolds (CP−) without SAL treatment (100% invasion). The asterisks represent significant differences between strain Newman treated and not treated with SAL (P = 0.0001) (A) and between Reynolds (CP5) organisms treated and not treated with SAL (P = 0.0089; Mann-Whitney test) (B). Comparison of Reynolds (CP5) treated with SAL and Reynolds (CP−) showed no significant differences.

FIG. 2.

Effect of SAL on cap5 (A), mgrA (B), and sae P3 (B) promoter activities. Expression of gfp driven by the target promoters was measured during the growth cycle, and fluorescence values were expressed as green fluorescent protein fluorescence related to the OD₆₅₀ (F/OD) in order to minimize variations in fluorescence due to varying cell density. The data represent the arithmetic means ± standard deviations for triplicate measurements from three or four independent experiments.

FIG. 3.

Real-time PCR analysis of cap, mgrA, and sae transcription. Changes in gene expression are shown as fold changes [−(ΔΔC_T)] following growth with 50 μg/ml of SAL to an OD₆₅₀ of 1.4. The data represent the means ± standard deviations for duplicate measurements from three independent experiments.

FIG. 4.

Immunodiffusion analysis of CP5 extracts. Thirty microliters of monospecific antiserum to serotype 5 was added to the center well. Thirty-microliter samples of undiluted and twofold serially diluted (indicated inside the well) CP5 extracts of Newman strain organisms, cultured or not with different concentrations of SAL, were added to the outer wells. Precipitin lines were visualized after staining with Coomassie brilliant blue. C+, positive control [undiluted extract of S. aureus Reynolds (CP5) strain]; C−, negative control [undiluted extract of S. aureus Reynolds (CP−) strain]. Precipitin lines were visible up to a 1:4 dilution of capsular extracts in the control without SAL, up to a 1:2 dilution at a 0.36 mM SAL concentration, and at only a 1:1 dilution at the highest SAL concentration (2 mM).