The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action

DISCUSSION

The concept of adaptive bacterial systems for sensing and responding to host cues to achieve immune avoidance or subversion is well established (16). Among these mechanisms, the GraSR multicomponent system appears to be integral to S. aureus defense against innate immune effector HDPs (6, 7). The current investigation builds upon our previous work by exploring the mechanisms of resistance mediated through GraS and/or its EL in response to HDPs of relevant immune context and under cognate physiologic conditions (3, 4, 17). Several key findings which shed new light on the role of GraSR in the pathogenic relationship of S. aureus with the human host emerged from these studies.

Overall, the present data established that wild-type and mutant S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5. This finding is consistent with our prior reports, demonstrating that S. aureus susceptibility to α-defensins and certain other HDPs is greater at pH 7.5 than at pH 5.5 (5, 13). Importantly, the current data revealed that distinct HDPs, even structurally related defensins of different immune compartments, exert unambiguously distinct actions against S. aureus. Furthermore, these effects appear to be pH dependent. For example, in the wild-type strain, hNP-1-mediated killing at either pH 7.5 or 5.5 correlated significantly with increases in ANX and SSC. These findings are consistent with our prior report showing that the HDP thrombin-induced platelet microbial protein 1 (tPMP-1) induces cytoplasmic membrane invagination and nucleic acid condensation correlating with chromosomal condensation, cytoplasmic refractivity, and inhibition of macromolecular synthesis (18, 19). hBD-2 exerted anti-S. aureus efficacy predominantly at pH 7.5. This efficacy was associated with significant hyperpolarization (ENR) and ANX in the wild-type strain. The staphylocidal effects of these mechanisms appear to be pH dependent, given that similar events observed at pH 5.5 were not associated with hBD-2 efficacy. In contrast, the platelet kinocidin mimetic RP-1 exerted multiple actions at both pH 7.5 and 5.5. The key mechanistic signature of RP-1 efficacy in wild-type S. aureus included simultaneous increases in SSC, PRM, ANX, and CDP, with a significant decrease in ENR. These mechanisms were amplified at pH 7.5 versus 5.5., consistent with greater RP-1 efficacy at pH 7.5. Importantly, the mechanistic signature of each HDP mentioned above was clearly distinct from that of the nonspecific detergent SDS. This observation affords cogent evidence that HDPs exert specific, targeted, and context-dependent effects that confront S. aureus and its GraSR system.

The ΔgraS and ΔEL mutants exhibited significant and equivalent increases in susceptibility to hNP-1 under pH conditions designed to reflect bloodstream (pH 7.5) or phagolysosomal (pH 5.5) immune contexts. Mechanistically, increases in hNP-1-induced killing of these mutants at pH 7.5 corresponded to decreased SSC and ANX compared to those for the wild-type strain. These results suggest that the GraS holoprotein confers adaptive responses to S. aureus that yield intracellular countermeasures manifesting as increased CM turnover, including changes in phospholipid location and/or composition. At pH 7.5, the finding that these events were equivalent in the EL and graS mutants strongly suggests that, at least for hNP-1, the EL is sufficient for the sensing function of the GraSR system. At pH 5.5, hNP-1 exhibited less efficacy than at pH 7.5 for all strains tested. However, the ΔgraS mutant exhibited greater susceptibility to hNP-1 at pH 5.5 than the wild type, and in inverse relation to ANX. Further, the ANX response was dependent on the GraS system, as both the ΔgraS and ΔEL mutants were deficient in mounting a protective ANX response observed in the wild-type parent. This theme was amplified at pH 5.5, a condition likely encountered within the neutrophil phagolysosome.

Several new insights also emerged regarding the interaction of hBD-2 with the GraS system in S. aureus. At pH 7.5, neither the ΔgraS nor the ΔEL mutation rendered S. aureus more susceptible to hBD-2. However, at pH 5.5, the ΔgraS mutation led to an increase in susceptibility to this HDP; a lesser effect was also observed with the ΔEL mutant. This pattern of results suggests novel intersections of GraS function and hBD-2 activity under distinct pH conditions. First, efficacy at pH 7.5 corresponded to impairment of ANX in the ΔgraS mutant, indicating that GraS mediates important CM turnover required for survival in response to hBD-2. Second, given that neither the charge nor the conformation of hBD-2 is significantly different at pH 7.5 versus 5.5 (13, 17), the GraSR system rather than the peptide hBD-2 may function more differently under distinct pH conditions. Third, the observation that the full ΔgraS deletion, and not the ΔEL mutation alone, conferred distinct vulnerability to hBD-2 indicates that the GraS holoprotein is more influenced by pH than is its EL. Several mechanisms underlying this relationship may exist, including conformational dynamics, the impact of a ΔpH gradient (electron motive force), or the influence of charge or osmotic conditions targeting the holoprotein rather than the EL sensor motif. Thus, it appears that at neutral pH (e.g., in the bloodstream), the holoprotein and the EL domain of the sensor are functionally equivalent. However, at acidic pH (e.g., in phagolysosomes), EL sensing of an HDP may not be as effectively transduced by the GraS holoprotein and thus may fail to convey an adaptive survival response(s) to the organism. This finding is supported by the observation that the sEL protected the ΔgraS mutant against hBD-2 at pH 5.5. Therefore, the GraS holoprotein and its EL motif appear to play complementary, nonredundant roles in sensing specific hBD-2 and conveying adaptive responses to S. aureus.

Compared to defensin hNP-1 or hBD-2, the platelet kinocidin mimetic peptide RP-1 exerted a much more complex mechanistic signature that correlated with overall greater susceptibility of S. aureus under pH conditions reflecting the bloodstream (pH 7.5) or phagolysosome (pH 5.5). The ΔgraS mutant, and to a lesser extent the ΔEL mutant, exhibited significant increases in key mechanisms of RP-1 action compared to controls. These mechanisms included increases in CM functions (increased PRM and decreased ENR) and the rapid induction of cell death-like pathway(s) (e.g., increased ANX and CDP) compared to the case for the wild type. Consistent with these findings, the sEL failed to protect any S. aureus strain from killing by RP-1 at either pH. Collectively, these findings indicate that RP-1 exerts multiple effects against which the GraS holoprotein or its EL afford protective responses. As RP-1 is not a defensin and differs markedly from hNP-1 or hBD-2 in physicochemical parameters, it is likely that HDPs of distinct structure exert differential actions against which the GraSR system may or may not convey effective resistance. Thus, the considerably greater efficacy compared to other study HDPs suggests that multiple S. aureus countermeasures are required for survival in response to RP-1 in the bloodstream or upon interactions of platelets with neutrophils in the acidic phagolysosome (3).

The current findings are consistent with prior data which suggested that GraSR is involved in stress responses and potential mechanisms of action of other peptide anti-infective agents, including daptomycin, LL-37, and the experimental agent brilacidin (20). Other studies have suggested that GraX as well as distinct two-component regulatory systems, such as VraSR and NsaSR, are also involved in S. aureus responses to antibiotics (21, 22). Likewise, Stk1/Stp1 signaling appears to cross talk with GraSR in modulating S. aureus cell wall charge (23). GraSR may have special relevance to sensing peptide-based agents, including vancomycin (glycopeptide), daptomycin (lipopeptide), and structurally or functionally similar host defense peptides.

In a broader view, the present results also support hypotheses regarding novel mechanisms by which peptide anti-infectives may target S. aureus via the GraSR or other systems (30–33). The current data suggest that certain HDPs induce cell death pathways in S. aureus such as exist in many other pathogenic bacteria (14, 15, 24–28). For example, when exposed to certain HDPs, S. aureus exhibits a sharp increase in annexin V binding (ANX) consistent with access to hydroxylated or otherwise negatively charged lipids (29). S. aureus is not known to make phosphatidylserine, the classic binding ligand of annexin V; whether a novel phosphatidylserine intermediate or like lipid emerges as a result of HDP exposure remains to be determined. Likewise, the present data suggest that certain peptides lead to activation of proteases associated with cell death (CDP) which cleave amino acid motifs of caspase- or metacaspase-like substrates. These findings align with responses to HPD actions reflecting pathways (14, 15) that are initiated by altered membrane integrity (e.g., permeability [PRM] and potential [ENR]), leading to chromosomal condensation (e.g., SSC [18; this study) and perturbed macromolecular synthesis (19) and yielding rapid and irreversible cell death (see Fig. S2 in the supplemental material). GraS-mediated countermeasures to these mechanisms appear to involve cell membrane potential (ENR) equilibration and membrane lipid turnover (ANX) to limit further HDP-mediated injury and minimize HDP access to intracellular targets. Thus, deficient GraS holoprotein or EL-mediated functions render S. aureus less able to adaptively respond to HDPs and more vulnerable to killing by certain HDPs. While HDP-mediated cell death in S. aureus has been understudied to date, the current findings suggest coordinated programs that have the potential to be targeted by novel anti-infective peptides or other agents.

Limitations of the current studies should also be understood. Most importantly, studying the effects of HDPs in vitro cannot fully recapitulate the complex conditions under which they function in vivo. In addition, only selected mechanistic parameters were studied in the current investigations. Although our novel approach to define mechanistic signatures led to new insights into HDP interactions with S. aureus, there are likely other mechanisms of HDP action that are counteracted by GraS/EL which contribute to differences in susceptibility under distinct conditions. Studies using increasingly specific GraS or EL mutations, broader GraSR TCS disruptions, and more specific mechanistic probes should afford a more complete understanding. Investigation of potentially unique structure-mechanism relationships of HDPs with the GraSR system is in progress in our laboratory. Nonetheless, the present results offer new insights into the host-pathogen relationships between HDPs and S. aureus adaptive resistance responses, which are likely more complex than has been understood previously.