The novel protein ScrA acts through the SaeRS two-component system to regulate virulence gene expression in Staphylococcus aureus

Abstract

Staphylococcus aureus is a Gram-positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two-component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureus clumping regulator A). To investigate the mechanism of action of ScrA we performed proteomics and transcriptomics in a ScrA overexpressing strain and show that a number of surface adhesins are upregulated, while secreted proteases are downregulated. Results also showed upregulation of the SaeRS TCS, suggesting that ScrA is influencing SaeRS activity. Overexpression of ScrA in a saeR mutant abrogates the clumping phenotype confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a positive regulator of scrA expression. Collectively, our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems.

Keywords: Staphylococcus aureus; SaeRS; small proteins; virulence.

FIGURE 1

Predicted transcript architecture of the scrAB locus. (a) Three overexpression plasmids were constructed to express either a his tagged ScrA (pScrA‐his), scrA (pScrA), or scrA and scrB (pScrAB). (b) Data previously published by Mäder et al. (2016) suggest that scrAB is encoded on a polycistronic transcript. (c) Northern blot of wild‐type S. aureus containing the pMK4 empty vector (WT pMK4), wild‐type S. aureus containing pscrAB (WT pScrAB), and the scrA mutant (scrA). Blots were probed with a riboprobe antisense to the scrA open reading frame. Blots were loaded with either 10 or 20 μg of total RNA as indicated. Red bar in panel a indicates the sequence used to generate the scrA northern probe, while the scrB probe sequence in indicated by a green bar

FIGURE 2

Overexpression of scrA induces clumping. (a) S. aureus containing the pMK4 empty vector as well as the three overexpression constructs were grown overnight and were left static for 2 h. The initial and final OD₆₀₀ of the top 100 μl were used to calculate clumping. (b) The scrAB overexpression plasmid was transduced into S. aureus strains SH1000, Newman, and UAMS‐1, and clumping was assessed. An increase in clumping was observed in each background. (c) Individual overexpression of either scrA, scrB, or scrAB was performed in plasmid pCN51 to determine the contribution of each individual transcript to clumping. Overexpression was driven by a cadmium‐inducible promoter. Increased clumping was only observable when overexpressing scrA. (d) A biofilm formation assay was performed over time with wild‐type S. aureus containing the pMK4 empty vector (pMK4) and the scrAB overexpressing strain (pScrAB). A statistically significant increase in biofilm formation was observed in the scrAB overexpressing strain. Experiments were performed for a minimum of three times for panels a, b, c, and d, respectively. Error bars represent standard deviation. Statistical significance was determined using an ordinary one‐way ANOVA and Tukey's multiple comparison for panels a–c. Student's t‐test was used at each time point for panel d; *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

FIGURE 3

Role of the ScrA protein in clumping. (a) Serum‐free clumping assay performed using S. aureus containing the empty vector (pCN51), overexpressing native ScrA (pCN51_ScrA), overexpressing ScrA with a nonsense mutation at amino acid 3 (pCN51_ScrA_NSAA3), or overexpressing ScrA with a nonsense mutation at amino acid 8 (pCN51_ScrA_NSAA8). (b) Serum‐free clumping assay performed on S. aureus containing either the empty vector (pCN51, pMK4), overexpressing full‐length scrA (pCN51_ScrAB, pMK4_ScrAB), overexpressing the ScrA C‐terminal tail (pCN51_ScrA‐CTD), or overexpressing the ScrA transmembrane domain (pMK4_ScrA‐TM). Experiments were performed for a minimum of three times. Error bars represent standard deviation. Statistical significance was determined using an ordinary one‐way ANOVA and Tukey's multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

FIGURE 4

Transcriptomic and proteomic analysis of ScrA overexpressing strain. (a) RNA sequencing was performed on 3 h cultures of S. aureus containing the pMK4 empty vector and the scrAB overexpressing strain. Differential expression analysis was performed, and the results were visualized on a volcano plot. Significance was determined using Student's t‐test. Log₂ fold change is shown on the x axis, while −log₁₀ p is shown on the y axis. Genes indicted by red circles displayed a fold change >2 and p value was <0.05. (b) The 10 genes demonstrating the highest fold increase and decrease in expression in the scrAB overexpressing strain. Several adhesions, including coa, empbp, and sbi, were identified as being upregulated in a scrAB overexpresser. (c) The secreted protein profiles of S. aureus containing the pMK4 empty vector and the scrAB overexpressing strain were analyzed by mass spectrometry proteomics. Differential expression analysis was performed, and the results were visualized on a volcano plot. Significance was determined using Student's t‐test. Log₂ fold change is shown on the x axis, while −log₁₀ p is shown on the y axis. Genes indicated by red circles displayed a fold change >2 and p value was <0.05. (d) the 10 proteins demonstrating the highest fold increase and decrease in abundance in the scrAB overexpressing strain

FIGURE 5

ScrAB overexpressing leads to increased hemolytic activity against human erythrocytes. Cultures of S. aureus containing the pMK4 empty vector (WT pMK4_EV) and the scrAB overexpressing strain (WT pScrAB) were grown for 15 h and hemolysis assays were performed with cell‐free culture supernatants. An ~7 fold increase in hemolytic activity was observed in the scrAB overexpressing strain compared to the empty vector control. Experiments were performed four times. Error bars represent standard deviation. Significance was determined using Student's t‐test *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

FIGURE 6

The SaeRS system is required for ScrA‐mediated phenotypes. (a) Clumping assays were performed in wild‐type S. aureus, saeR, and saeS mutants overexpressing scrAB. No increase in clumping was observed when either saeR or saeS was disrupted. (b) Biofilm assays were performed in wild‐type S. aureus and an saeR mutant overexpressing scrAB. No increase in biofilm formation was observed when ScrA was overexpressed in the saeR mutant. Experiments were performed four times. Error bars represent standard deviation. Significance was determined using a standard one‐way ANOVA and Tukey's multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

FIGURE 7

ArlR positively regulates scrA expression. (a) Reanalysis of data previously published by Rapun‐Araiz et al. (2020) demonstrated that constitutive expression of ArlR resulted in an approx. 25‐fold increase in scrA expression. No other TCS response regulator increased scrA expression when constitutively activated. (b) Quantification of scrA transcript abundance in S. aureus WT, arlR, and saeR mutant strains containing the scrAB overexpression plasmid following 3, 6, and 9 h growth. Abundance of scrA transcript in each strain was determined by RT‐qPCR and normalized against the value in the WT strain at each timepoint. A significant decrease in scrA expression was observed in the arlR mutant at both 3 and 6 h, but there was no significant difference by 9 h. Interestingly disruption of saeR resulted in increased scrA expression at 3, 6, and 9 h of growth. (c) Clumping assays were performed using WT S. aureus and the arlR mutant containing the scrAB overexpression plasmid. Overexpressing scrAB in the arlR mutant led to a significant increase in clumping, but not to the same extent observed in the WT strain. The increase in clumping in the arlR mutant was significantly lower than that in the WT background. Experiments were performed four times. Error bars represent standard deviation. Significance was determined using a Student's t‐test for panel B and an ordinary one‐way ANOVA and Tukey's multiple comparison for panel C. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

FIGURE 8

Overexpression of scrAB leads to membrane instability in an SaeRS‐dependent manner. Propidium iodide staining was used to measure membrane stability. Increased fluorescence is indicative of greater instability. Overexpression of scrAB in the wild‐type strain resulted in a significant decrease in membrane stability, while no difference in membrane stability was observed following scrAB overexpression in the saeR mutant strain. Membrane instability was also increased following scrAB overexpression in the arlR mutant although not to the same degree as in WT S. aureus. Error bars represent standard deviation. Significance was determined using an ordinary one‐way ANOVA and Tukey's multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

FIGURE 9

Hypothetical working model of ArlRS‐ScrA‐SaeRS action. ArlR positively regulates scrA expression, either directly or indirectly. The ScrA protein inserts into the cell membrane which in turn activates the SaeRS system, causing increased expression of Sae regulon genes. The increase in Sae system activity has a negative feedback on scrA expression either directly through SaeR or indirectly via a downstream Sae regulon gene