Rat/MgrA, a regulator of autolysis, is a regulator of virulence genes in Staphylococcus aureus

Abstract

We have previously identified mgrA (rat) as a regulator of autolysis in Staphylococcus aureus. Besides its effect on autolytic activity, we recently found alterations in the expression of regulator and target virulence genes in the mgrA mutant. Northern analysis and transcription fusion assays showed that inactivation of mgrA has led to the downregulation of RNAIII of agr and hla and upregulation of sarS and spa. Although both SarA and agr are activators of alpha-hemolysin and a repressors of protein A synthesis, we found that the transcription of sarA was not affected in the mgrA mutant and vice versa, indicating that MgrA likely regulates hla and spa in a SarA-independent manner. Previously we have shown that SarT, a SarA homolog, is a repressor of hla and an activator of spa, presumably by activating SarS, however, analysis of the double sarT mgrA mutant for hla and spa transcription indicated that the mgrA-mediated effect is not mediated via sarT. Our results further demonstrated that the mgrA gene product regulates hla and spa expression in a dual fashion, with the first being agr dependent and the second agr independent. In the agr-independent pathway, MgrA binds directly to hla and the sarS promoter to modulate alpha-hemolysin and protein A expression. Thus, our studies here have defined the nature of interaction of mgrA with other regulators such as agr, sarS, and sarT and its role in regulating hla and spa transcription within the virulence regulatory network of S. aureus.

FIG. 1.

Effect of the mgrA mutation on the expression of genes involved in virulence. A. Northern blot analysis of hla and spa transcripts in RN6390 and isogenic mgrA mutant ALC2530. The open reading frame of each gene was used as a probe for the Northern blots. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. The expression of GFP driven by the hla and spa promoters in RN6390 and the mgrA mutant was also measured. Promoter activation was plotted as mean fluorescence/OD₆₅₀ ratio, using average values of triplicate readings at an OD₆₅₀ of ≈1.4. Comparable differences were also observed at earlier points. These experiments were repeated at least three times with similar results. ▧, RN6390; ░⃞, mgrA mutant ALC2530. B. Northern blot analysis of sarA and saeRS transcripts in RN6390 and mgrA mutant ALC2530. The open reading frame of each gene was used as a probe for the Northern blots. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. C. Northern blot analysis of agr RNAIII and sarS transcripts in RN6390 and mgrA mutant ALC2530. The probe for RNAIII was a 920-bp fragment containing the hld gene. The open reading frame of the sarS gene was used as a probe for the Northern blot. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. The expression of GFP driven by the agr RNAIII and sarS promoters in RN6390 and the mgrA mutant was also measured. Promoter activation was plotted as mean fluorescence/OD₆₅₀ ratio, using average values of triplicate readings at an OD₆₅₀ of ≈1.4. Comparable differences were also observed at earlier points. These experiments were repeated at least three times with similar results. ▧, (RN6390; ░⃞, mgrA mutant ALC2530).

FIG. 2.

Transcription of agr RNAIII and hla in RN6390 and isogenic mutants. A. Northern blot analysis of agr RNAIII transcripts in RN6390 and isogenic mutants. The probe for RNAIII was a 920-bp fragment containing the hld gene. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. The expression of GFP driven by the agr RNAIII (P3) promoter was also measured. Promoter activation was plotted as mean fluorescence/OD₆₅₀ ratio, using average values of triplicate readings at an OD₆₅₀ of ≈1.4. This experiment was repeated at least three times with similar results. ▧, RN6390; ░⃞, mgrA mutant ALC2530; ▥, sarT mutant ALC3043; ▩, agr mutant RN6911; ▤, sarT mgrA mutant ALC3046; , agr mgrA mutant ALC2537; ▪ agr sarT mutant ALC3188; and □ mgrA sarT agr mutant ALC3191. B. Northern blot analysis of the hla transcript in RN6390 and isogenic mutants. The probe for hla was an 800-bp fragment encompassing the open reading frame. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. The expression of GFP driven by the hla promoter was also measured. Promoter activation was plotted as mean fluorescence/OD₆₅₀ ratio, using average values of triplicate readings at an OD₆₅₀ of ≈1.4. This experiment was repeated at least three times with similar results. ▧, RN6390; ░⃞, mgrA mutant ALC2530; ▥, sarT mutant ALC3043; ▩, agr mutant RN6911; ▤, sarT mgrA mutant ALC3046; , agr mgrA mutant ALC2537; ▪ agr sarT mutant ALC3188; and □ mgrA sarT agr mutant ALC3191.

FIG. 3.

Gel shift assays of purified MgrA with hla and agr promoter fragments. MgrA protein in increasing concentrations was incubated with end-labeled hla or agr promoter fragments. In competition assays, MgrA protein (1,500 ng) was incubated with the promoter fragment in the presence of a 50-fold excess of unlabeled specific competitor (hla or agr) (lane 7) or nonspecific competitors (≈300-bp asp23 promoter in lane 8 and ≈160-bp spa promoter fragment in lane 9).

FIG. 4.

Transcription of hla by providing RNAIII in trans. Northern blot analysis of agr RNAIII and hla transcripts in strains with and without plasmid RN6735. (Transformants are indicated as strain/Pl.) RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase.

FIG. 5.

Transcription of sarS in RN6390 and isogenic mutants. A. Northern blot analysis of the sarS transcript in RN6390 and isogenic mutants. The open reading frame of sarS was used as a probe. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. B. The expression of GFP driven by the sarS promoter was also measured. Promoter activation was plotted as mean fluorescence/OD₆₅₀ ratio, using average values of triplicate readings at an OD₆₅₀ of ≈1.4. This experiment was repeated at least three times with similar results. ▧, RN6390; ░⃞, mgrA mutant ALC2530; ▥, sarT mutant ALC3043; ▩, agr mutant RN6911; ▤, sarT mgrA mutant ALC3046; , agr mgrA mutant ALC2537; ▪ agr sarT mutant ALC3188; and □ mgrA sarT agr mutant ALC3191.

FIG. 6.

Transcription of spa in RN6390 and isogenic mutants. A. Northern blot analysis of the spa transcript in RN6390 and isogenic mutants. The open reading frame of spa was used as a probe. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase. B. The expression of GFP driven by the spa promoter was also measured. Promoter activation was plotted as mean fluorescence/OD₆₅₀ ratio, using average values of triplicate readings at an OD₆₅₀ of ≈1.4. This experiment was repeated at least three times with similar results. ▧, RN6390; ░⃞, mgrA mutant ALC2530; ▥, sarT mutant ALC3043; ▩, agr mutant RN6911; ▤, sarT mgrA mutant ALC3046; , agr mgrA mutant ALC2537; ▪ agr sarT mutant ALC3188; and □ mgrA sarT agr mutant ALC3191.

FIG. 7.

Gel shift assays of purified MgrA with sarS promoter fragment. MgrA protein in increasing concentrations was incubated with the end-labeled sarS promoter fragment. In competition assays, MgrA protein (1,000 ng) was incubated with end-labeled promoter in the presence of a 50-fold excess of unlabeled specific competitor (sarS) (lane 7) or the nonspecific competitor (≈300-bp asp23 promoter in lane 8 and the ≈160-bp spa promoter fragment in lane 9).

FIG. 8.

Transcription of spa by providing RNAIII in trans. Northern blot analysis of the spa transcript in strains with and without plasmid RN6735. (Transformants are indicated as strain/Pl.) The open reading frame of the spa gene was used as a probe. RNA was harvested from cells grown to an OD₆₅₀ of ≈1.2, representing late exponential phase.

FIG. 9.

Model for MgrA-mediated bimodal regulation of virulence genes in S. aureus. MgrA upregulates expression of RNAIII. RNAIII activates hla expression and downregulates sarS, leading to repression of spa. Besides the agr-dependent pathway, MgrA binds directly to the hla promoter to augment its activation. Similarly, MgrA binds to the sarS promoter to downregulate spa expression.