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 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 acid, but not palmitoleic or sapienic acid. However, enhanced resistance to these C16:1 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 extra-cytoplasmic 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% 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.

GraXRS-Dependent Resistance of Staphylococcus aureus to Human Osteoarthritic Synovial Fluid

“…This work highlights the critical importance of the GraXRS regulatory system and the Gra-regulated mprF gene as a resistance system against innate immune effectors. Previous work has shown that GraS is able to sense acidic pH, such as that of human skin, or the phagolysosome of macrophages and respond through GraR-mediated upregulation of resistance genes, such as mprF , allowing for bacterial survival ( 29 , 31 , 43 ). However, as stated previously, it is known that synovial fluid from osteoarthritis patients has a neutral pH of approximately 7.35 ( 14 ), so GraS must sense some factor or condition, possibly CAMPs, in the synovial fluid other than the pH in order to respond through GraR-mediated gene expression.…”

Two-Component Systems of S. aureus: Signaling and Sensing Mechanisms

“…Direct interaction of the EL loop region (DYDFPIDSL) with CAMPs is also likely in S. aureus , since a soluble EL mimic of GraS protected S. aureus against CAMP-mediated death [ 53 ]. The proline at position 39 and the two aspartic acid residues at positions 37 and 41 located within the EL are important for full GraS activity [ 53 , 54 , 55 ].…”

“…Recent work has expanded the sensory capabilities of GraS to include the sensing of acidic pH [ 54 , 55 , 59 ], and GraS activation at low pH is mandatory for survival within macrophages [ 58 ]. Interestingly, pH-dependent modulation of GraS activity seems to be independent of the Gra-EL implicated in CAMP sensing [ 58 ].…”

“…Acidic pH reduces the charge on polar lipid head groups, which presumably impacts GraS activity. It was hypothesized that acidic pH should also reduce the charge on acidic amino acids in the EL of GraS, which would render them less effective in sensing CAMP at low pH [ 55 ]. An EL-independent mechanism of GraS activation under acidic conditions might be beneficial for the bacterium.…”

Identification and Application of a Panel of Constitutive Promoters for Gene Overexpression in Staphylococcus aureus

“…The xylose-inducible promoter system is repressible by glucose ( Wieland et al, 1995 ), which also prohibits its use in in vivo studies, as glucose is a common constituent inside mammalian cells. The tetracycline-inducible promoter system is currently the most functional inducible system for gene expression in S. aureus ( Bateman et al, 2001 ; Harrison et al, 2019 ; Goncheva et al, 2020 ; Kuiack et al, 2020 ). However, the inducers such as tetracycline or anhydrotetracycline required in this system, in our experience, affect the normal metabolism of or are even toxic to S. aureus , which results in undesirable phenotypes.…”