Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix

Abstract

The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm.IMPORTANCEStaphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Keywords: CodY; PIA; Staphylococcus aureus; biofilm; eDNA; exopolysaccharide; lipoproteins.

FIG 1

ΔcodY mutant cells of diverse S. aureus clinical isolates form large cell aggregates tethered by a stringlike matrix. (A) Scanning electron microscopy was performed on SA564 and ΔcodY mutant cells during exponential growth in tryptic soy broth. Images are representative of multiple experiments. Images are at the same magnification. Representative images of biofilm observed in overnight culture growth are shown to the left of each micrograph. (B and C) Percent aggregation of S. aureus clinical isolates and their ΔcodY mutant derivatives (B) and the complemented SA564 codY-null mutant (C) using the settling assay from samples obtained during exponential growth in TSB as described in Materials and Methods. Data indicate the mean ± standard error of the mean (SEM) values from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (relative to WT using Student’s t test [B] and analysis of variance [ANOVA] with Dunnett’s postanalysis relative to SA564 WT [C]). VOC, vector-only control.

FIG 2

ΔcodY mutant cell aggregates are associated with extracellular DNA (eDNA) and are sensitive to DNase I treatment. (A) Cells were grown to exponential phase in TSB, stained with Syto 40 and TOTO-1, and then visualized using confocal scanning laser microscopy (CSLM). Live cells are blue (Syto 40), while eDNA and dead cells are green (TOTO-1). Each panel is viewed at the same magnification. Insets are at ×10 magnification. Asterisks indicate the areas used for insets. (B) SEM micrographs of SA564 cells grown aerobically to exponential phase in TSB containing DNase I. All images are shown at the same magnification. (C) Settling assay of cells grown in TSB aerobically to exponential phase in shake flask culture. (D) Static biofilm development in the presence of increasing amounts of DNase I was measured at 20 h after inoculation in TSB as described in Materials and Methods. Data are the mean ± SEM values from at least three independent experiments. **, P < 0.01; ****, P < 0.0001 (relative to wild type using Student’s t test [C] or ANOVA with Dunnett’s postanalysis [relative to wild type, 0 U/ml] [D]). NS, not significant. All images are representative of multiple experiments.

FIG 3

eDNA-based ΔcodY mutant cell aggregation is protease tolerant and initially dependent on electrostatic interactions. (A) The effect of proteinase K (0.1 mg ml⁻¹) on wild-type and ΔcodY mutant cells of SA564 was examined during exponential growth in TSB using a settling assay. (B) A settling assay was performed on exponentially growing cells cultured in TSB buffered to pH 7.5 or unbuffered TSB, which acidified to ∼pH 6.5 during growth. (C) Cells cultured in TSB were resuspended in phosphate-buffered saline at either pH 5.0 or 7.5 for 15 min, and quantitative PCR was used to measure the amount of eDNA released from the samples, taken at the same OD₆₀₀ value. Representative micrographs are shown for ΔcodY mutant cells from the same experiment. Syto 40 (blue signal) was used to stain all cells, while eDNA was visualized using TOTO-1 (green signal). Data are the mean ± SEM values from at least three independent experiments. *, P < 0.05; ***, P < 0.001 (by Student’s t test comparing ΔcodY to wild type [A and B] or Friedman’s test with Dunn’s postanalysis [C]). Here, results for the wild-type cell sample suspended in PBS pH 7.5 are significantly different from those for the ΔcodY mutant cell sample at pH 5.0 and 7.5 and trended higher than those for the wild type at pH 5.0 (but were not significantly different).

FIG 4

Coculture experiments reveal that eDNA-based aggregates consist of predominantly ΔcodY mutant cells. Cultures were inoculated with the indicated genotypes at approximately a 1:1 ratio, grown to exponential phase (OD₆₀₀ of ∼0.5) in TSB, and then visualized by CSLM. All cells are labeled with Syto 40 (blue); eDNA and dead cells are labeled with TOTO-1 (green). In each panel a particular strain harbors a constitutive dsRed plasmid (pKM16) to discern genotypes. (A) SA564 wild-type cells mixed with isogenic ΔcodY mutant cells harboring pKM16. (B) SA564 wild-type cells harboring pKM16 mixed with isogenic ΔcodY mutant cells. (C) Nonaggregating LAC ΔcodY mutant cells mixed with SA564 ΔcodY mutant cells harboring pKM16. (D) SA564 ΔcodY mutant cells mixed with aggregating COL ΔcodY mutant cells harboring pKM16. All images are representative of multiple experiments. All panels are viewed at the same magnification.

FIG 5

Suppressor analysis reveals that ica is required for cell aggregation. (A) SRB1243 cells (SA564 ΔcodY soa-1) were grown to exponential phase in TSB and assayed for aggregation. ****, P < 0.0001 (by ANOVA with Dunnett’s postanalysis comparing samples to wild type). (B) SEM of suppressor mutant cells sampled during exponential growth in tryptic soy broth. The image is representative of multiple experiments. (C) Settling assay performed on isogenic strains during exponential growth in TSB. **, P < 0.01; ***, P < 0.001 (by ANOVA with Dunnett’s postanalysis). Here, the ΔcodY and mraY* ΔcodY mutants are significantly different from the wild type. (D) Complementation using the sarA P1 promoter to constitutively express icaB. ***, P < 0.001 (by two-tailed Student t test comparing +icaB [P_sarAP1-icaB⁺] to +vector). (E) CSLM micrographs of ΔcodY soa-1 cells harboring pCN51 or pKM26 (P_sarAP1-icaB⁺) during exponential growth in TSB. All cells were visualized using Syto 40 (blue signal), while eDNA and dead cells were stained by TOTO-1 (green signal). All images are representative of multiple experiments. All panels are viewed at the same magnification.

FIG 6

eDNA-based cell aggregation is dependent on the production of PIA in ΔcodY mutant cells. (A) SA564 and LAC cells were grown to exponential phase aerobically in TSB, and icaA transcript copy numbers in wild-type and ΔcodY mutant cells were determined by qRT-PCR. Data were normalized to rpoC transcript copy number. (B and C) Quantification of cell-associated PIA detected by immunoblot analysis using densitometry for SA564 or isogenic mutants (B) or the wild type and codY-null mutant of the indicated strains (C) obtained from cell pellets grown aerobically for 3 h in tryptic soy broth. When necessary, samples were diluted to avoid membrane saturation. (D) SA564 and ΔcodY mutant cells were grown aerobically in TSB containing sodium metaperiodate (40 μg ml⁻¹) or dispersin B (5 μg ml⁻¹), and aggregation was assessed using the settling assay. (E) Wild-type SA564 and LAC cells constitutively expressing icaADBC under the control of the sarA P1 promoter were grown planktonically in tryptic soy broth, and a settling assay was used to assess aggregation. SA564 was additionally cultured in the presence of DNase I (200 U ml⁻¹). Data indicate the mean ± SEM values from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (using Student’s t test comparing the ΔcodY mutant to the wild type for each condition in panels A, D, and E). ns, not significantly different. Error bars are plotted for all data; in some cases, they are too small to see.

FIG 7

PIA and bacterial chromosomal DNA promote cell aggregation in CDM. Wild-type SA564 cells containing pKM28 (P_sarA-P1-icaADBC) or the vector-only control (pMRSI) were cultured in CDM lacking exogenous DNA. During exponential growth, a 1-ml sample of cells was mixed with purified genomic DNA from S. aureus LAC (A), Bacillus subtilis SMY (B), or Pseudomonas aeruginosa PAO1 (C), and a settling assay was performed. Data are plotted as the mean ± SEM values from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by one-way ANOVA with Dunnett’s posttest comparing samples to SA564 + pMRSI [0 ng/ml DNA]).

FIG 8

Cell wall-anchored lipoproteins contribute to eDNA/PIA adherence to the cell surface. (A) An isogenic suite of SA564 mutant strains were grown in TSB, and a settling assay was performed as described in Materials and Methods. **, P < 0.01; ***, P < 0.001 (compared to the wild-type and Δlgt strains. #, P < 0.05 (comparing the ΔcodY mutant to the Δlgt ΔcodY double mutant). One-way ANOVA with Tukey postanalysis was used. SEMs are plotted for all data; in some cases the error bars are too small to see. (B) Immunoblot densitometry analysis of PIA production for the indicated strains is shown. When necessary, samples were diluted to avoid membrane saturation.

FIG 9

Working model of PIA/eDNA-dependent cell aggregation. (A) As the abundance of key nutrients (i.e., branched-chain amino acids and GTP) drops intracellularly, CodY activity decreases, promoting cell aggregation using available eDNA and PIA. (B) Cell-to-cell interaction occurs in a CodY-dependent manner whereby eDNA and PIA interact synergistically with the cell surface, mediated by one or more lipoproteins. eDNA, blue threads; PIA, red polygons; lipoproteins, green ovals.