Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

Abstract

Background: Staphylococcus epidermidis (SE) has emerged as one of the most important causes of nosocomial infections. The SaeRS two-component signal transduction system (TCS) influences virulence and biofilm formation in Staphylococcus aureus. The deletion of saeR in S. epidermidis results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in S. epidermidis remains unclear.

Results: The saeRS genes of SE1457 were deleted by homologous recombination. The saeRS deletion mutant, SE1457ΔsaeRS, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction) by DNase I treatment compared to SE1457 and the complementation strain SE1457saec. Compared to SE1457 and SE1457saec, SE1457ΔsaeRS showed increased Triton X-100-induced autolysis (approximately 3-fold) and decreased cell viability in planktonic/biofilm states; further, SE1457ΔsaeRS also released more extracellular DNA (eDNA) in the biofilms. Correlated with the increased autolysis phenotype, the transcription of autolysis-related genes, such as atlE and aae, was increased in SE1457ΔsaeRS. Whereas the expression of accumulation-associated protein was up-regulated by 1.8-fold in 1457ΔsaeRS, the expression of an N-acetylglucosaminyl transferase enzyme (encoded by icaA) critical for polysaccharide intercellular adhesin (PIA) synthesis was not affected by the deletion of saeRS.

Conclusions: Deletion of saeRS in S. epidermidis resulted in an increase in biofilm-forming ability, which was associated with increased eDNA release and up-regulated Aap expression. The increased eDNA release from SE1457ΔsaeRS was associated with increased bacterial autolysis and decreased bacterial cell viability in the planktonic/biofilm states.

Figure 1

Effect of DNaseI on SE1457ΔsaeRS, SE1457, and SE1457saec biofilm formation. SE1457ΔsaeRS, SE1457, and SE1457saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P < 0.05. WT, SE1457; SAE, SE1457ΔsaeRS; SAEC, SE1457saec.

Figure 2

SEM and TEM observations of SE1457ΔsaeRS and wild-type strain. (A) Biofilms of SE1457ΔsaeRS, SE1457, and SE1457saec after 24 h of growth on hydroxyapatite disks were observed by SEM. Arrows show the extracellular polymeric substances (EPSs) (10,000× magnification). (B) Planktonic cells of SE1457ΔsaeRS and SE1457 cultured for 24 h were observed by TEM. Cell-cell accumulations in SE1457ΔsaeRS are circled; arrow indicates the thread-like material linking neighboring cells. WT, SE1457; SAE, SE1457ΔsaeRS; SAEC, SE1457saec.

Figure 3

Effect of saeRS deletion on Triton X-100-induced autolysis. SE1457ΔsaeRS, SE1457, and SE1457saec cells were diluted in TSB medium containing 1 M NaCl, grown to mid-exponential phase (OD600 = ~0.6-0.8), and resuspended in the same volume of 0.05 M Tris-HCl solution containing 0.05% Triton X-100 (pH 7.2). OD600 readings were measured every 30 min. The autolysis rate induced by Triton X-100 was calculated as follows: lysis rate = OD0 - ODt/OD0. The experiments were carried out in triplicate independently. WT, SE1457; SAE, SE1457ΔsaeRS; SAEC, SE1457saec.

Figure 4

Zymographic analysis of autolytic enzyme extracts. Bacteriolytic enzyme profiles were analyzed on SDS gels (10% separation gel) containing lyophilized M. luteus cells (0.2%) or S. epidermidis cells (0.2%) as substrates. After electrophoresis, the gels were washed for 30 min in distilled water, incubated for 6 h at 37°C in a buffer containing Triton X-100, and then stained with methylene blue. The S. epidermidis atlE mutant was used as a negative control. Bands with lytic activity were observed as clear zones in the opaque gel. The clear zones appeared as dark bands after photography against a dark background. The molecular mass standard is shown on the left of the gels. Ex-Lys, cell-wall extracts of lysostaphin-treated S. epidermidis; Ex-SDS, cell-wall extracts of SDS-treated S. epidermidis; Ex-Sup, concentrated S. epidermidis culture supernatants; WT, SE1457; SAE, SE1457ΔsaeRS; SAEC, SE1457saec; ATLE, SE1457ΔatlE.

Figure 5

Viability of S. epidermidis 1457 in biofilms and the planktonic state. (A) CFU counts of SE1457ΔsaeRS and SE1457. After 0, 6, 12, and 24 h of incubation, CFUs for SE1457 and SE1457ΔsaeRS cultures were calculated using serial dilutions of each sample plated on 6 agar plates. (B) CLSM images of S. epidermidis biofilms. SE1457 and SE1457ΔsaeRS were incubated in glass-bottomed cell culture dishes. After incubation at 37°C for 24 h, SE1457ΔsaeRS and SE1457 cells in biofilms were stained with LIVE/DEAD reagents that indicate viable cells by green fluorescence (SYTO9) and dead cells by red fluorescence (PI). Results depict a stack of images taken at approximately 0.3 μm depth increments and represent one of the three experiments. Fluorescence intensities were quantified using ImageJ software. WT, SE1457; SAE, SE1457ΔsaeRS.

Figure 6

Quantification of eDNA in SE1457ΔsaeRS, SE1457, and SE1457saec biofilms. eDNA was extracted from the unwashed 24 h biofilms of SE1457ΔsaeRS (white bars), SE1457 (black bars), and SE1457saec (gray bars). The eDNA in each biofilm was quantified by qPCR using primers specific for gyrA, serp0306, lysA, and leuA [19,28]. The quantity of eDNA was calculated as follows: total eDNA (ng)/relative OD600. Results represent the mean ± SD of three independent experiments. WT, SE1457; SAE, SE1457ΔsaeRS; SAEC, SE1457saec.

Figure 7

S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I. (A) Attached SE1457ΔatlE, SE1457ΔsaeRS, SE1457 and SE1457saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P < 0.05; WT, SE1457; SAE, SE1457ΔsaeRS; SAEC, SE1457 saec; ATLE, SE1457ΔatlE.