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Novel Functions and Signaling Specificity for the GraS Sensor Kinase of Staphylococcus aureus in Response to Acidic pH.

ABSTRACT: Although the GraS sensor kinase of Staphylococcus aureus is known for the sensing of and resistance to cationic antimicrobial peptides (CAMPs), we recently established that it also signals in response to acidic pH, which is encountered on human skin concurrently with CAMPs, antimicrobial unsaturated free fatty acids (uFFA), and calcium. We therefore evaluated how these environmental signals would affect GraS function and resistance to antimicrobial uFFA. Growth at pH 5.5 promoted increased resistance of S. aureus USA300 to linoleic and arachidonic acids but not palmitoleic or sapienic acid. However, enhanced resistance to these C_16:1 uFFA was achieved by supplementing acidic medium with 0.5 mM calcium or subinhibitory CAMPs. Enhanced resistance to uFFA at acidic pH was dependent on GraS and GraS-dependent expression of the lysyl-phosphatidylglycerol synthase enzyme MprF, through a mechanism that did not require the lysyl-transferase function of MprF. In addition to enhanced resistance to antimicrobial uFFA, acidic pH also promoted increased production of secreted proteases in a GraS-dependent manner. During growth at pH 5.5, downstream phenotypes of signaling through GraS, including resistance to uFFA, MprF-dependent addition of positive charge to the cell surface, and increased production of secreted proteases, all occurred independently of acidic amino acids in the extracytoplasmic sensor loop of GraS that were previously found to be required for sensing of CAMPs. Cumulatively, our data indicate that signaling through GraS at acidic pH occurs through a mechanism that is distinct from that described for CAMPs, leading to increased resistance to antimicrobial uFFA and production of secreted proteases.IMPORTANCE Staphylococcus aureus asymptomatically colonizes 30% of humans but is also a leading cause of infectious morbidity and mortality. Since infections are typically initiated by the same strain associated with asymptomatic colonization of the nose or skin, it is important to understand how the microbe can endure exposure to harsh conditions that successfully restrict the growth of other bacteria, including a combination of acidic pH, antimicrobial peptides, and antimicrobial fatty acids. The significance of our research is in showing that acidic pH combined with antimicrobial peptide or environmental calcium can signal through a single membrane sensor protein to promote traits that may aid in survival, including modification of cell surface properties, increased resistance to antimicrobial fatty acids, and enhanced production of secreted proteases.

SUBMITTER: Kuiack RC

PROVIDER: S-EPMC7585063 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacterial two-component systems are crucial features of bacterial pathogens such as methicillin-resistant Staphylococcus aureus to overcome environmental and antimicrobial stresses by activating regulons to interfere with the bactericidal mechanisms. GraRS is a unique subset of two-component systems belonging to the intramembrane-sensing histidine kinase family (IM-HK) and is responsible for resistance to cationic host defense peptides. However, the precise manner by which the short 9-residue extracellular loop of the membrane sensor GraS detects the antimicrobial peptides and transduces the signal is not comprehensively understood. Here, we show that a single point mutation (D35A) in the extracellular loop of GraS blocked activation of GraRS, but this effect was also abrogated with graS mutations in the N-terminal transmembrane segments without any accompanying effect on GraS protein expression. Additionally, mutations in H120 and T172 in the dimerization/histidine phosphotransfer (DHp) domain of GraS increased activation without any accompanying enhancement in dimerization, likely due to disruption of the H120-T172 interaction that restricts rotational movements of the DHp helices since swapping H120 and T172 did not alter GraS activation. Notably, the enhancing effects of H120 and T172 mutations were abolished with a D35 mutation, highlighting the pivotal role of D35 in the 9-residue extracellular loop of GraS in GraR phosphorylation. In summary, our study delivers the significance of the D35 in the extracellular loop of GraS and ensuing changes in the N-terminal transmembrane helices as a model to illustrate signaling in the IM-HK subset of two-component regulatory systems. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen capable of infecting skin, blood, internal organs, and artificial medical devices. Generally, personal hygiene and a robust immune system can limit the spread of this pathogen; however, MRSA possesses an assortment of phenotypic tools to survive the hostile host environment including host defense peptides. More specifically, S. aureus utilizes two-component systems to sense noxious environmental cues to respond to harmful environmental elements. Our study focused on a two-component system called GraRS that S. aureus deploys against host defense peptides. We showed that one single residue in the extracellular loop of GraS and the adjacent membrane segment controlled the activation of GraRS, indicating the importance of a well-tuned-charged residue in the extracellular loop of GraS for sensing activity.

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Novel Functions and Signaling Specificity for the GraS Sensor Kinase of Staphylococcus aureus in Response to Acidic pH.

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