Transcriptional downregulation of agr expression in Staphylococcus aureus during growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC

Abstract

The temporal and cell density-dependent regulation of expression of virtually all the Staphylococcus aureus virulon is under the control of the agr (accessory gene regulatory) operon. The expression of the agr operon is subject to transcriptional regulation by the AgrA/C two-component response regulator/sensor kinase pair. During bacteraemia, a frequent syndrome caused by methicillin-resistant S. aureus (MRSA), the transcriptional downregulation of agr expression has been attributed to the sequestration of the quorum-signalling molecule auto-inducing peptide (AIP) by the human serum component apolipoprotein B as part of an innate immune response to infection. However, it is not known whether transcriptional downregulation of agr expression during growth in human serum is additionally subjected to regulation by transcription regulatory proteins that either directly or indirectly affect transcription from the agr operon promoters. Here, using chromosomal fluorescence reporters of agr expression in S. aureus, we show that the transcriptional downregulation of agr expression in human serum can be overcome using constitutive active mutant forms of AgrC. Therefore, it seems that the sequestration of the AIP is likely to be the only mechanism by which the host innate immune response limits agr expression at the transcriptional level to maintain the host-pathogen balance towards a noninvasive outcome.

Keywords: GFP transcriptional reporters; transcription regulation; two-component systems.

Figure 1

(a) Schematic representation of the agr operon organisation and regulation in Staphylococcus aureus. (b) Schematic representation showing single-site chromosomal integration of GFP transcriptional fusion reporters for P2 and P3. (c) Sequence of P2 and P3 promoter regions used to generate the gfp transcriptional fusions for P2 and P3 (the consensus −10 and −35 sites are outlined, the transcription start sites are shown with arrows and the RBS is underlined. (d) Sheep blood agar haemolysis assay with S. aureus USA300_WT (1), USA300_agrA::Tn [agrA transposon mutant from NARSA library (Fey et al., 2013)] (2), USA300_{agr IR P3-GFP} (3) and USA300_{agr IR P2-GFP} (4). (e) Graphs showing relative hld, agrA and gyrB mRNA levels in 16 h cultures of S. aureus USA300_WT and USA300_{agr IR P3-GFP} as determined by Taqman qRT-PCR. Values are shown for each gene with respect to USA300_WT levels. (f) Graph showing growth curves (OD₆₀₀) of S. aureus USA300_WT, USA300_{agr IR P2-GFP} and USA300_{agr IR P3-GFP} strains grown in TSB. (g) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] and GFP-FU as a function of growth (OD₆₀₀) over time for S. aureus USA300_{agr IR P2-GFP} and USA300_{agr IR P3-GFP} strains grown in TSB. (h) As in (g) but with S. aureus SH1000_{agr IR P2-GFP}, SH1000_{agr IR P3-GFP}, SH1001_{agr IR P2-GFP} and SH1001_{agr IR P3-GFP} strains grown in TSB. Data for (d–h) were obtained from three biological replicates

Figure 2

(a) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD₆₀₀) over time for Staphylococcus aureus USA300_{agr IR P2-GFP} and USA300_{agr IR P3-GFP} strains grown in TSB media containing 0–50% (v/v) human serum. (b) Graph showing GFP expression (as GFP fluorescence units (GFP-FU)) as a function of growth (OD₆₀₀) over time for USA300_{agr IR P2-GFP} and USA300_{agr IR P3-GFP} strains grown in TSB media upon addition of 25% (v/v) human serum to late-exponentially growing cells (indicated with a green arrow). (c) As in (b), however, readings for OD₆₀₀ and GFP expression values were taken every 30 min before the addition of human serum and every 10 min thereafter. Data for graphs (a–c) were obtained from three biological replicates.

Figure 3

Western blot indicating intracellular AgrA levels in Staphylococcus aureus strain USA300_WT during growth in TSB medium in the absence and presence of human serum and/or tetracycline. A schematic representation of the experimental steps is shown on the top of the blot (see text for details). Samples of whole-cell extracts were prepared for the analysis immediately before and 2 and 16 h after the addition of human serum (T₀, T₂ and T₁₆ respectively). Lane 8 contains purified recombinant HMK-tagged AgrA as a positive control marker (Reynolds & Wigneshweraraj, 2011). Data shown are representative from three independent experiments (biological repeats). AgrA signal intensity values were calculated by densitometry using a Typhoon FLA 7000 and indicated relative to the signal intensity at T₀.

Figure 4

Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] as a function of growth (OD₆₀₀) over time for Staphylococcus aureus USA300_{agr IR P3-GFP} strain containing plasmids encoding for mutant and wild-type forms of AgrC (as indicated) grown in TSB with 25% (v/v) human serum added after cells reached late-exponential growth phase (indicated with a green arrow). Data shown were obtained from three biological replicates.