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. 2018 Mar 26;200(8):e00012-18.
doi: 10.1128/JB.00012-18. Print 2018 Apr 15.

Nutritional Regulation of the Sae Two-Component System by CodY in Staphylococcus aureus

Kevin D Mlynek  1 , William E Sause  2 , Derek E Moormeier  3 , Marat R Sadykov  3 , Kurt R Hill  1 , Victor J Torres  2 , Kenneth W Bayles  3 , Shaun R Brinsmade  4
Affiliations

Nutritional Regulation of the Sae Two-Component System by CodY in Staphylococcus aureus

Kevin D Mlynek et al. J Bacteriol. .

Abstract

Staphylococcus aureus subverts innate defenses during infection in part by killing host immune cells to exacerbate disease. This human pathogen intercepts host cues and activates a transcriptional response via the S. aureus exoprotein expression (SaeR/SaeS [SaeR/S]) two-component system to secrete virulence factors critical for pathogenesis. We recently showed that the transcriptional repressor CodY adjusts nuclease (nuc) gene expression via SaeR/S, but the mechanism remained unknown. Here, we identified two CodY binding motifs upstream of the sae P1 promoter, which suggested direct regulation by this global regulator. We show that CodY shares a binding site with the positive activator SaeR and that alleviating direct CodY repression at this site is sufficient to abrogate stochastic expression, suggesting that CodY represses sae expression by blocking SaeR binding. Epistasis experiments support a model that CodY also controls sae indirectly through Agr and Rot-mediated repression of the sae P1 promoter. We also demonstrate that CodY repression of sae restrains production of secreted cytotoxins that kill human neutrophils. We conclude that CodY plays a previously unrecognized role in controlling virulence gene expression via SaeR/S and suggest a mechanism by which CodY acts as a master regulator of pathogenesis by tying nutrient availability to virulence gene expression.IMPORTANCE Bacterial mechanisms that mediate the switch from a commensal to pathogenic lifestyle are among the biggest unanswered questions in infectious disease research. Since the expression of most virulence genes is often correlated with nutrient depletion, this implies that virulence is a response to the lack of nourishment in host tissues and that pathogens like S. aureus produce virulence factors in order to gain access to nutrients in the host. Here, we show that specific nutrient depletion signals appear to be funneled to the SaeR/S system through the global regulator CodY. Our findings reveal a strategy by which S. aureus delays the production of immune evasion and immune-cell-killing proteins until key nutrients are depleted.

Keywords: CodY; Sae; Sae TCS; Staphylococcus aureus; branched-chain amino acids; gene regulation; two-component system; virulence.

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Figures

FIG 1
FIG 1
CodY and Sae control virulence gene expression. (A) Schematic of the saePQRS locus. The green boxes at promoters denote SaeR binding sites. The weak but constitutive P3 promoter drives expression of saeRS; the P1 promoter is inducible and drives expression of saePQRS. (B) SaeS responds to multiple stimuli (yellow), and this promotes autophosphorylation of SaeS and phosphotransfer to SaeR. SaeR∼P can then bind its various targets. (C) Regulatory relationships between Agr, Rot, CodY, and Sae. Major global regulators under CodY control and known to affect sae P1 promoter activity are shown. The dashed lines indicate a potential indirect mechanism of CodY-mediated repression; the solid line indicates potential direct regulation. Question marks indicate an unknown or postulated regulatory mechanism.
FIG 2
FIG 2
CodY regulates saePQRS in a Rot-dependent and Rot-independent manner. Mean transcript abundances are shown for the indicated strains during exponential growth (OD600 of ∼0.5) in TSB medium using qRT-PCR. saeP (A) and nuc (B) transcripts were normalized to rpoC transcript in two independent lineages of S. aureus, UAMS-1 and USA300 LAC. Statistical significance for each condition was assessed using one-way ANOVA and Tukey's multiple comparison posttest (*, P < 0.05; **, P < 0.01; N.S., not significant). Dotted lines denote significance for the indicated background and strains. Error bars represent the standard errors of the means from at least two independent experiments. When not visible, error bars are too small to see and are obscured by the bars.
FIG 3
FIG 3
S. aureus CodY interacts with the sae P1 upstream region. (A) The region surrounding the sae P1 regulatory region is displayed (coding strand; 5′ to 3′). The known translated sequence, transcriptional start site (+1), −10, and −35 sites are displayed and annotated in boldface. The identified SaeR∼P footprint is bracketed with the direct and indirect repeat motifs indicated above the sequence with a dotted line. A dashed line above the sequence identifies the putative CodY motifs. mm, mismatch. (B) Purified SaCodY-His6 protein was incubated with a 6-FAM-labeled 235-bp DNA fragment containing the upstream regulatory region of saeP in the presence of ILV and GTP. The unbound fragment is indicated with open arrows while CodY-DNA complexes are indicated by filled arrows. Increasing amounts of CodY monomer were incubated with the fragment. The molar concentration (of monomeric protein) used in each reaction mixture is indicated above each lane. (C) CodY monomer (120 nM) was incubated with the fragment in the presence of a 30× molar excess of unlabeled probe. The presence of each protein and unlabeled probe is indicated beneath each lane.
FIG 4
FIG 4
DNase I footprinting reveals that the CodY and SaeR binding sites overlap. A 5′-FAM-labeled saeP235p+ fragment incubated in the presence of 0, 60, and 120 nM purified CodY or bovine serum albumin (BSA; control) was challenged with 0.075 to 0.1 U of DNase I. The resulting fragments were separated using capillary electrophoresis and aligned to the sequenced PCR product as a reference. Relative fluorescence (y axis) is displayed as a function of nucleotide position (x axis). Reaction mixtures containing CodY (gray trace) were compared to control reaction mixtures containing bovine serum albumin (black trace). A drop in peak intensity at a given nucleotide position indicates protection by CodY. CodY binds to a 31-bp region (colored nucleotides). Data are representative of at least two independent experiments.
FIG 5
FIG 5
CodY contributes to the stochastic expression pattern of saeP. Flow cytometry analysis was performed of UAMS-1 cells harboring either the saeP235p+-gfp (A) or saeP235pscrambled-gfp (B) reporter fusions during exponential growth in TSB medium. Histograms indicate the relative number of cells (y axis) exhibiting a given fluorescence intensity (x axis) and are scaled to 30,000 cells to account for various sample sizes. Data are the means ± the standard errors of the means of at least two independent experiments.
FIG 6
FIG 6
codY mutant cells express thermonuclease (nuc) at early stages of S. aureus biofilm development. S. aureus (UAMS-1) wild-type and codY mutant cells containing the nuc-gfp reporter plasmid pMRSI-nuc were grown in the BioFlux 1000 microfluidic system. Bright-field and epifluorescence microscopic images were acquired at a magnification of ×200 after 6 h of biofilm development. Images are representative of multiple experiments.
FIG 7
FIG 7
CodY-enhanced PMN killing is dependent on the Sae TCS. Primary human PMNs were intoxicated with supernatants obtained from codY null mutants at either 3 h (A) or 5 h (B) following subculture. Intoxication experiments were performed on two independent occasions, with a total of four blood donors. Statistical analysis was performed by ANOVA with Dunnett's multiple-comparison test. Asterisks indicate P values for comparisons between results for the wild type (LAC) and the codY null mutant (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent the standard errors of the means.
FIG 8
FIG 8
Working model of how nutritional regulation controls SaeR/S. CodY integrates the nutritional status of the cell with host cues at the sae P1 promoter. Under conditions of nutrient excess, sae P1 transcription is repressed. As nutrients become depleted, CodY repression is lifted, eventually resulting in an increase in SaeR/S activity and the expression of exoproteins to damage host tissue. This allows S. aureus to scavenge nutrients from the host environment.
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