Staphylococcus aureus AI-2 quorum sensing associates with the KdpDE two-component system to regulate capsular polysaccharide synthesis and virulence

Abstract

Autoinducer 2 (AI-2) is widely recognized as a signal molecule for intra- and interspecies communication in Gram-negative bacteria, but its signaling function in Gram-positive bacteria, especially in Staphylococcus aureus, remains obscure. Here we reveal the role of LuxS in the regulation of capsular polysaccharide synthesis in S. aureus NCTC8325 and show that AI-2 can regulate gene expression and is involved in some physiological activities in S. aureus as a signaling molecule. Inactivation of luxS in S. aureus NCTC8325 resulted in higher levels of transcription of capsular polysaccharide synthesis genes. The survival rate of the luxS mutant was higher than that of the wild type in both human blood and U937 macrophages. In comparison to the luxS mutant, a culture supplemented with chemically synthesized 4,5-dihydroxy-2,3-pentanedione (DPD), the AI-2 precursor molecule, restored all the parental phenotypes, suggesting that AI-2 has a signaling function in S. aureus. Furthermore, we demonstrated that the LuxS/AI-2 signaling system regulates capsular polysaccharide production via a two-component system, KdpDE, whose function has not yet been clarified in S. aureus. This regulation occurred via the phosphorylation of KdpE binding to the cap promoter.

FIG. 1.

Growth and extracellular AI-2 production. Cultures of S. aureus NCTC8325 wild type (WT) and the luxS mutant (SX1) were inoculated into LB medium at time zero and at the indicated intervals. Growth was monitored, and aliquots were taken and filtered to remove cells. AI-2 activity in the supernatant was measured using the V. harveyi BB170 bioassay. The data shown represent the mean values of four parallel AI-2 bioassays. The fold induction of AI-2 in the wild type exceeded 100 when the OD₆₀₀ was 1.8.

FIG. 2.

Comparative measurement of transcription of CP synthesis-related genes. The relative transcript levels of the CP synthesis-related genes were determined in the S. aureus NCTC 8325 wild type (WT), the luxS mutant (SX1), and the luxS mutant with 39 nM AI-2 complementation (SX1 + AI-2). Strains were grown in LB medium with shaking at 37°C to an OD₆₀₀ of 2.1. AI-2 was added to the luxS mutant at the inoculation time to a final concentration of 39 nM. The RNA was extracted and transcription was quantified by real-time RT-PCR for the CP synthesis-related genes (represented by capA, capC, capE, capG, capI, capK, and capN). The quantity of cap cDNA measured by real-time PCR was normalized to the abundance of 16S cDNA within each reaction mixture. Error bars indicate the variance between triplicate samples within the real-time PCR. The relative cDNA abundance in the wild-type samples was arbitrarily assigned a value of 1.

FIG. 3.

Transcriptional regulation of the cap gene and kdpDE gene expression by LuxS/AI-2. (A) Relative transcript levels of the cap gene (represented by capN and capG). (B) Relative transcript levels of the kdpD, kdpE, and kdpA genes. The levels of transcription of these genes were measured by real-time RT-PCR in the S. aureus NCTC8325 wild type (WT), the luxS mutant (SX1), the luxS mutant with a plasmid containing luxS for genetic complementation (SX2), and the luxS mutant with 3.9 nM to 39 μM AI-2 for complementation (A1, 3.9 nM; A2, 39 nM; A3, 390 nM; A4, 3.9 μM; A5, 39 μM).

FIG. 4.

Transcriptional regulation of CP synthesis by KdpDE. The relative transcript levels of CP synthesis-related genes (represented by capA, capC, capE, capG, capI, capK, and capN) were measured by real-time RT-PCR in the S. aureus NCTC8325 wild type (WT), the kdpDE mutant (SX8), and the kdpDE mutant with a plasmid containing kdpDE (SX9).

FIG. 5.

Transcriptional regulation of cap gene expression by KdpE. (A) Relative transcript levels of the cap gene (represented by capN and capG) in the S. aureus NCTC8325 wild type (WT), the kdpDE mutant (SX8), the kdpDE mutant with a plasmid containing kdpDE (SX9), the kdpE mutant (SX10), the kdpE mutant with a plasmid containing kdpE (SX11), and the kdpE mutant with a plasmid encoding KdpE with a mutated Asp-phosphorylation site (SX12). (B) Binding of KdpE protein to the promoter of cap. The ability of KdpE to bind to the cap promoter was determined by gel-shift assay. Increasing amounts of KdpE were incubated with excess DIG-labeled probes. Lanes 1 to 6, KdpE concentrations of 0, 0.5, 1, 2, 4, and 4 μM, respectively; the amount of DIG-labeled probe in each case was 50 fmol (the concentration was 5 nM). In lane 6, besides the labeled probes, 500 fmol of unlabeled probes was incubated with the KdpE protein.

FIG. 6.

Alteration of survival of S. aureus in human blood and in U937 monocytic cells due to absence of luxS or kdpDE. (A) Rates of survival for the S. aureus NCTC8325 wild type (WT), the luxS mutant (SX1), the luxS mutant with a plasmid containing luxS (SX2), the luxS mutant with 39 nM AI-2 (SX1 + AI-2), the kdpDE mutant (SX8), and the kdpDE mutant with a plasmid containing kdpDE (SX9). The strains were incubated in heparinized human blood, and the results are from five separate blood donors. (B) S. aureus survival when it was cultured with U937 monocytic cells. The percentage of S. aureus CFU that survived was determined as described in Materials and Methods.

FIG. 7.

The QS systems in S. aureus. The Agr QS system regulates many virulence-associated traits in S. aureus, and the LuxS/AI-2 QS system exhibits clear involvement in CP production.