Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system

Abstract

The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation.

FIG. 1.

Biofilm formation ability of natural isolates of S. aureus in HHWm. (A) Biofilm formation by S. aureus strains on microtiter plates in HHWm and TSBg after 24 h of incubation at 37°C. The results of a representative experiment are shown. (B) Quantification of the biofilm formation capacity. The bacterial cells were stained with crystal violet and were quantified by dissolving the dye in ethanol-acetone (80:20), and the absorbance was determined at 595 nm. Data represent the averages of results from 12 wells from three different plates. The vertical line at the top of the each bar represents a standard deviation.

FIG. 2.

Biofilm formation phenotype of biofilm-positive mutants on microtiter plates in HHWm. (A) Biofilm formation phenotype of wild-type 15981 and isogenic mutants in all the TCS on microtiter plates grown in HHWm at 37°C for 24 h (Table 3). (B) Biofilm formation phenotype of wild-type and selected biofilm-positive mutants on microtiter plates grown in HHWm at 37°C for 24 h. Tn917 was inserted in different positions of the arlS gene in mutants MM4, -7, -13, and -14 and two different positions of the arlR gene in MM11 and MM19. (C) Biofilm formation phenotype of the ΔarlRS mutant compared to the wild-type strain and insertional transposon mutants. (D) Biofilm formation phenotype of the wild-type and ΔarlRS mutant strains complemented with either the parlRS plasmid carrying the arlRS operon under the control of its own promoter or the pCU1 plasmid. HHWm was supplemented with 20 μg/ml of chloramphenicol. The results of a representative experiment are shown.

FIG. 3.

Biofilm formation phenotype of the wild-type and ΔarlRS strains in microfermentors. (A) Biofilm development of bacteria grown under continuous-flow conditions with HHWm after 24 h at 37°C. The microfermentors (upper panels) contain the glass slides where bacteria form the biofilm (lower panels). The results of a representative experiment are shown. (B) Quantification of the biofilms adhered to the glass slides. The cells were removed from the glass slides into 10 ml of HHWm by using a vortex, and the OD of the resulting solution was measured at 650 nm. Significant differences were detected between the wild-type and the ΔarlRS mutant (n = 3; P < 0.05, Mann-Whitney U test).

FIG. 4.

Effect of an arlRS mutation on biofilm architecture. S. aureus 15981 and 15981 ΔarlRS strains harboring plasmid pSB2019 to provide constitutive GFP expression were grown in a four-well Lab-Tek II chamber with HHWm at 37°C for 24 h. (A) Biofilm development was monitored by scanning confocal laser microscope. The panels on the left are an overlay of multiple slices, and the side frames of the panels on the right are the z planes, which show the thickness of the biofilm and the architecture as viewed from the cross section. The line on the z plane indicates the level at which the photograph of the x-y plane was taken. Photographs were taken at a magnification of ×600. The results of a representative experiment are shown. (B) Quantification of the biofilm thickness using EZ-C1 software. Data represent the averages of results from 27 different fields from three independent experiments. The vertical line at the top of each bar represents the standard deviation. The asterisk represents a statistically significant difference (P < 0.0001, Student's t test).

FIG. 5.

(A) Dot blot analysis of PNAG accumulation in the wild-type strain and its corresponding ΔarlRS mutant at different points of the growth curve. Cell surface extracts at different times were treated with proteinase K, and dilutions of the extracts (1/2, 1/5, and 1/25) were spotted onto nitrocellulose membranes. PNAG production was detected with an anti-S. aureus PNAG antiserum. The ΔarlRS mutant produced higher levels of PNAG product at all points of the growth curve. The results of a representative experiment are shown. (B) Biofilm formation phenotype of the 15981, ΔarlRS, Δica, and ΔarlRS Δica strains grown in TSBg and HHWm on microtiter plates. After 24 h of incubation, the microplates were washed and stained with crystal violet. The dye was dissolved by the addition of 200 μl of ethanol-acetone (80:20). The results of a representative experiment are shown. (C) Biofilm detachment assays. Biofilms of the 15981, ΔarlRS, Δica, and ΔarlRS Δica strains grown in HHWm for 16 h were treated for 2 h at 37°C with 10 mM sodium metaperiodate or with 100 μg of proteinase K or pronase. The bacteria that remained attached to the surface were stained with crystal violet. The dye was dissolved by the addition of 200 μl of ethanol-acetone (80:20). The results of a representative experiment are shown. ON, overnight; WT, wild type.

FIG. 6.

Effects of the mutation of sarA and agr regulators on the biofilm promoted by an arlRS mutation. Bacteria were inoculated onto microtiter plates containing TSBg and HHWm. After 24 h of incubation, the microplates were washed and stained with crystal violet. The dye was dissolved in 200 μl of ethanol-acetone (80:20). The results of a representative experiment are shown. (A) Comparison of biofilm formations by S. aureus 15981 and ISP479r and their corresponding ΔarlRS, ΔsarA, and ΔarlRS ΔsarA mutants in HHWm and TSBg. (B) Comparison of biofilm formations by S. aureus strains 15981, 15981 ΔarlRS, ISP479r, and ISP479r's corresponding mutant derivatives ISP479r ΔarlRS, ISP479r-agr, and ISP479r-agr ΔarlRS in HHWm and TSBg. WT, wild type.

FIG. 7.

Effects of the mutation of atl on the biofilm promoted by an arlRS mutation. Comparison of biofilm formations by strain ISP479r and its corresponding ISP479r ΔarlRS, ISP479r-atl, and ISP479r ΔarlRS-atl mutant derivatives and by the same mutants constructed in the ISP479r agr null background. Bacteria were inoculated onto microtiter plates containing TSBg and HHWm. After 24 h of incubation, the microplates were washed and stained with crystal violet. The dye was dissolved in 200 μl of ethanol-acetone (80:20). The results of a representative experiment are shown.