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Rsp activates expression of the Cnt system in Staphylococcus aureus.

ABSTRACT: BACKGROUND:The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. RESULTS:A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. CONCLUSIONS:Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

SUBMITTER: Vinue L

PROVIDER: S-EPMC7594338 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Rsp activates expression of the Cnt system in Staphylococcus aureus.

Vinué Laura L Hooper David C DC

BMC microbiology 20201028 1

<h4>Background</h4>The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene.<h4>Results</h4>A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts ...[more]

PMID: 33115405

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Project description:Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States, an effect compounded by increasing antibiotic resistance. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects. We hypothesized that these effects are in part regulated by Notch signaling, for which ADAM10 activation is essential. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial and perivascular cells. Thus, we hypothesized that Hla would activate Notch in vascular cells. Human umbilical vein endothelial cells were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), or Hank's solution (HBSS), and probed by different methods. Luciferase assays showed that Hla (0.01 µg/mL) increased Notch activation by 1.75?±?0.5-fold as compared to HBSS controls (p?<?0.05), whereas Hla-H35L had no effect. Immunocytochemistry and Western blotting confirmed these findings and revealed that ADAM10 and ?-secretase are required for Notch activation after inhibitor and siRNA assays. Retinal EC in mice engineered to express yellow fluorescent protein (YFP) upon Notch activation demonstrated significantly greater YFP intensity after Hla injection than controls. Aortic rings from Notch reporter mice embedded in matrix and incubated with rHla or Hla-H35L demonstrate increased Notch activation occurs at tip cells during sprouting. These mice also had higher skin YFP intensity and area of expression after subcutaneous inoculation of S. aureus expressing Hla than a strain lacking Hla in both EC and pericytes assessed by microscopy. Human liver displayed strikingly higher Notch expression in EC and pericytes during S. aureus infection by immunohistochemistry than tissues from uninfected patients. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in vascular cells, an effect which may contribute to the pathobiology of infection with this microorganism.

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Rsp activates expression of the Cnt system in Staphylococcus aureus.

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Rsp activates expression of the Cnt system in Staphylococcus aureus.

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