Regulation of neutrophil myeloperoxidase inhibitor SPIN by the small RNA Teg49 in Staphylococcus aureus.

Teg49 is a Staphylococcus aureus trans-acting regulatory sRNA derived from cleavage of the sarA P3 transcript. We showed by RNA-Seq here that the 5' trident-like structure in Teg49 regulates transcriptionally (direct and indirect) 22 genes distinct from sarA. Among these, Teg49 was noted to repress spn, encoding a 102 residue preprotein which yields the mature 73 residue peptide which inhibits the catalytic activity of myeloperoxidase in human neutrophils. Teg49 was found to regulate spn mRNA post-transcriptionally in strain SH1000 through 9-nt base-pairing between hairpin loop 2 of Teg49 and an exposed bulge of the spn mRNA. Mutations of the Teg49 binding site disrupted the repression of spn, leading to reduced degradation, and increased half-life of spn mRNA in the Teg49 mutant. The spn-Teg49 interaction was also confirmed with a synonymous spn mutation to yield enhanced spn expression in the mutant vs. the parent. The Teg49 mutant with increased spn expression exhibited enhanced resistance to MPO activity in vitro. Killing assays with human neutrophils showed that the Teg49 mutant was more resistant to killing after phagocytosis. Altogether, this study shows that Teg49 in S. aureus has a distinct and important regulatory profile whereby this sRNA modulates resistance to myeloperoxidase-mediated killing by human neutrophils.

The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus.

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

GraS Sensory Activity in Staphylococcus epidermidis Is Modulated by the "Guard Loop" of VraG and the ATPase Activity of VraF.

Antimicrobial peptides (AMPs) are one of the key immune responses that can eliminate pathogenic bacteria through membrane perturbation. As a successful skin commensal, Staphylococcus epidermidis can sense and respond to AMPs through the GraXRS two-component system and an efflux system comprising the VraG permease and VraF ATPase. GraS is a membrane sensor known to function in AMP resistance through a negatively charged, 9-residue extracellular loop, which is predicted to be linear without any secondary structure. An important question is how GraS can impart effective sensing of AMPs through such a small unstructured sequence. In this study, we verified the role of graS and vraG in AMP sensing in S. epidermidis, as demonstrated by the failure of the ΔgraS or ΔvraG mutants to sense. Deletion of the extracellular loop of VraG did not affect sensing but reduced survival with polymyxin B. Importantly, a specific region within the extracellular loop, termed the guard loop (GL), has inhibitory activity since sensing of polymyxin B was enhanced in the ΔGL mutant, indicating that the GL may act as a gatekeeper for sensing. Bacterial two-hybrid analysis demonstrated that the extracellular regions of GraS and VraG interact, but interaction appears dispensable to sensing activity. Mutation of the extracellular loop of VraG, the GL, and the active site of VraF suggested that an active detoxification function of VraG is necessary for AMP resistance. Altogether, we provide evidence for a unique sensory scheme that relies on the function of a permease to impart effective information processing. IMPORTANCE Staphylococcus epidermidis has become an important opportunistic pathogen that is responsible for nosocomial and device-related infections that account for considerable morbidity worldwide. A thorough understanding of the mechanisms that enable S. epidermidis to colonize human skin successfully is essential for the development of alternative treatment strategies and prophylaxis. Here, we demonstrate the importance of an AMP response system in a clinically relevant S. epidermidis strain. Furthermore, we provide evidence for a unique sensory scheme that would rely on the detoxification function of a permease to effect information processing.

Role of the Staphylococcus aureus Extracellular Loop of GraS in Resistance to Distinct Human Defense Peptides in PMN and Invasive Cardiovascular infections.

GraS is a membrane sensor in Staphylococcus aureus that induces mprF and dltABCD expression to alter the surface positive charge upon exposure to cationic human defense peptides (HDPs). The sensing domain of GraS likely resides in the 9-residue extracellular loop (EL). In this study, we assessed a hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strain (COL) for the specific role of two distinct EL mutations: F38G (bulk) and D/35/37/41K (charged inversion). Activation of mprF by polymyxin B (PMB) was reduced in the D35/37/41K mutant versus the D35/37/41G mutant, correlating with reduced surface positive charge; in contrast, these effects were less prominent in the F38G mutant but still lower than those in the parent. These data indicated that both electrostatic charge and steric bulk of the EL of GraS influence induction of genes impacting HDP resistance. Using mprF expression as a readout, we confirmed GraS signaling was pH dependent, increasing as pH was lowered (from pH 7.5 down to pH 5.5). In contrast to PMB activation, reduction of mprF was comparable at pH 5.5 between the P38G and D35/37/41K point mutants, indicating a mechanistic divergence between GraS activation by acidic pH versus cationic peptides. Survival assays in human blood and purified polymorphonuclear leukocytes (PMNs) revealed lower survival of the D35/37/41K mutant versus the F38G mutant, with both being lower than that of the parent. Virulence studies in the rabbit endocarditis model mirrored whole blood and PMN killing assay data described above. Collectively, these data confirmed the importance of specific residues within the EL of GraS in conferring essential bacterial responses for MRSA survival in infections.

CspA regulation of Staphylococcus aureus carotenoid levels and σB activity is controlled by YjbH and Spx.

Staphyloxanthin, a carotenoid in S. aureus, is a powerful antioxidant against oxidative stresses. The crtOPQMN operon driving pigment synthesis is under the control of σB . CspA, a cold shock protein, is known to control σB activity. To ascertain genes that regulate cspA, we screened a transposon library that exhibited reduced cspA expression and pigmentation. We found that the adaptor protein YjbH activates cspA expression. Spx, the redox-sensitive transcriptional regulator and a proteolytic target for YjbH and ClpXP, complexes with αCTD of RNAP prior to binding the cspA promoter to repress cspA activity. Increased cspA expression in trans in the inactive spx C10A mutant of JE2 did not enhance pigment production while it did in JE2, suggesting that cspA is downstream to Spx in pigmentation control. As the staphyloxanthin pigment is critical to S. aureus survival in human hosts, we demonstrated that the cspA and yjbH mutants survived less well than the parent in whole blood killing assay. Collectively, our studies suggest a pathway wherein YjbH and ClpXP proteolytically cleave Spx, a repressor of cspA transcription, to affect σB -dependent carotenoid expression, thus providing a critical link between intracellular redox sensing by Spx and carotenoid production to improve S. aureus survival during infections.

YpdA, a putative bacillithiol disulfide reductase, contributes to cellular redox homeostasis and virulence in Staphylococcus aureus.

The intracellular redox environment of Staphylococcus aureus is mainly buffered by bacillithiol (BSH), a low molecular weight thiol. The identity of enzymes responsible for the recycling of oxidized bacillithiol disulfide (BSSB) to the reduced form (BSH) remains elusive. We examined YpdA, a putative bacillithiol reductase, for its role in maintaining intracellular redox homeostasis. The ypdA mutant showed increased levels of BSSB and a lower bacillithiol redox ratio vs. the isogenic parent, indicating a higher level of oxidative stress within the bacterial cytosol. We showed that YpdA consumed NAD(P)H; and YpdA protein levels were augmented in response to stress. Wild type strains overexpressing YpdA showed increased tolerance to oxidants and electrophilic agents. Importantly, YpdA overexpression in the parental strain caused an increase in BSH levels accompanied by a decrease in BSSB concentration in the presence of stress, resulting in an increase in bacillithiol redox ratio vs. the vector control. Additionally, the ypdA mutant exhibited decreased survival in human neutrophils (PMNs) as compared with the parent, while YpdA overexpression protected the resulting strain from oxidative stress in vitro and from killing by human neutrophils ex vivo. Taken together, these data present a new role for YpdA in S. aureus physiology and virulence through the bacillithiol system.

α-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus-Infected Wounds.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1ß secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1ß promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

Small RNA teg49 Is Derived from a sarA Transcript and Regulates Virulence Genes Independent of SarA in Staphylococcus aureus.

Expression of virulence factors in Staphylococcus aureus is regulated by a wide range of transcriptional regulators, including proteins and small RNAs (sRNAs), at the level of transcription and/or translation. The sarA locus consists of three overlapping transcripts generated from three distinct promoters, all containing the sarA open reading frame (ORF). The 5' untranslated regions (UTRs) of these transcripts contain three separate regions ∼711, 409, and 146 nucleotides (nt) upstream of the sarA translation start, the functions of which remain unknown. Recent transcriptome-sequencing (RNA-Seq) analysis and subsequent characterization indicated that two sRNAs, teg49 and teg48, are processed and likely produced from the sarA P3 and sarA P1 transcripts of the sarA locus, respectively. In this report, we utilized a variety of sarA promoter mutants and cshA and rnc mutants to ascertain the contributions of these factors to the generation of teg49. We also defined the transcriptional regulon of teg49, including virulence genes not regulated by SarA. Phenotypically, teg49 did not impact biofilm formation or affect overall SarA expression significantly. Comparative analyses of RNA-Seq data between the wild-type, teg49 mutant, and sarA mutant strains indicated that ∼133 genes are significantly upregulated while 97 are downregulated in a teg49 deletion mutant in a sarA-independent manner. An abscess model of skin infection indicated that the teg49 mutant exhibited a reduced bacterial load compared to the wild-type S. aureus Overall, these results suggest that teg49 sRNA has a regulatory role in target gene regulation independent of SarA. The exact mechanism of this regulation is yet to be dissected.

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.

Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections.

Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

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

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous ß-defensin (human ß-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.

Dysregulation of mprF and dltABCD expression among daptomycin-non-susceptible MRSA clinical isolates.

BACKGROUND: In small series or individual reports, SNPs within the mprF ORF and dysregulation of its expression in Staphylococcus aureus have been linked to daptomycin resistance (DAP-R) via a proposed gain-in-function mechanism. Similarly, dysregulation of dltABCD has also been associated with DAP-R. METHODS: Using 22 well-characterized, isogenic daptomycin-susceptible (DAP-S)/DAP-R clinical MRSA strain pairs, we assessed potential relationships of the DAP-R phenotype with: (i) regulation of mprF transcription; (ii) regulation of dltABCD transcription; (iii) expression of the two-component regulatory system, graRS (upstream regulator for both mprF and dltABCD transcription); (iv) SNPs within the graRS promoter or its ORF; and (v) altered mprF transcription and lysyl-phosphatidylglycerol (L-PG) synthesis. RESULTS: Enhanced expression of mprF occurred with SNPs in highly distinct and well-chronicled MprF domain 'hot spots' and rarely occurred without such mutations. Increased expression and/or dysregulation of mprF and dltABCD were not uncommon in DAP-R strains, occurring in 27% of strains for each gene. In these latter strains, neither graRS expression profiles nor polymorphic sequences within the graRS promoter or ORF could be significantly linked to altered transcription of mprF or dlt. CONCLUSIONS: Although graRS can co-regulate mprF and dltABCD expression, loci outside of this regulon appear to be involved in dysregulation of these latter two genes and the DAP-R phenotype. Finally, DAP-R strains exhibiting significantly altered mprF transcription profiles produced significantly increased levels of L-PG.

Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance.

Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.

Characterization of a novel small molecule that potentiates ß-lactam activity against gram-positive and gram-negative pathogens.

In a loss-of-viability screen using small molecules against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 with a sub-MIC of a ß-lactam, we found a small molecule, designated DNAC-1, which potentiated the effect of oxacillin (i.e., the MIC of oxacillin decreased from 64 to 0.25 µg/ml). Fluorescence microscopy indicated a disruption in the membrane structures within 15 min of exposure to DNAC-1 at 2× MIC. This permeabilization was accompanied by a rapid loss of membrane potential, as monitored by use of the DiOC2 (3,3'-diethyloxacarbocyanine iodide) dye. Macromolecular analysis showed the inhibition of staphylococcal cell wall synthesis by DNAC-1. Transmission electron microscopy of treated MRSA USA300 cells revealed a slightly thicker cell wall, together with mesosome-like projections into the cytosol. The exposure of USA300 cells to DNAC-1 was associated with the mislocalization of FtsZ accompanied by the localization of penicillin-binding protein 2 (PBP2) and PBP4 away from the septum, as well as mild activation of the vraRS-mediated cell wall stress response. However, DNAC-1 does not have any generalized toxicity toward mammalian host cells. DNAC-1 in combination with ceftriaxone is also effective against an assortment of Gram-negative pathogens. Using a murine subcutaneous coinjection model with 10(8) CFU of USA300 as a challenge inoculum, DNAC-1 alone or DNAC-1 with a sub-MIC of oxacillin resulted in a 6-log reduction in bacterial load and decreased abscess formation compared to the untreated control. We propose that DNAC-1, by exerting a bimodal effect on the cell membrane and cell wall, is a viable candidate in the development of combination therapy against many common bacterial pathogens.

Role of adaptor TrfA and ClpPC in controlling levels of SsrA-tagged proteins and antitoxins in Staphylococcus aureus.

Staphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus.

Site-specific mutation of the sensor kinase GraS in Staphylococcus aureus alters the adaptive response to distinct cationic antimicrobial peptides.

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.

Neutrophil-derived IL-1ß is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1ß/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1ß at the site of infection. Furthermore, neutrophil-derived IL-1ß was essential for host defense since adoptive transfer of IL-1ß-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1ß-deficient mice. S. aureus-induced IL-1ß production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1ß and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1ß production by neutrophils.

In vivo bioluminescence imaging to evaluate systemic and topical antibiotics against community-acquired methicillin-resistant Staphylococcus aureus-infected skin wounds in mice.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) frequently causes skin and soft tissue infections, including impetigo, cellulitis, folliculitis, and infected wounds and ulcers. Uncomplicated CA-MRSA skin infections are typically managed in an outpatient setting with oral and topical antibiotics and/or incision and drainage, whereas complicated skin infections often require hospitalization, intravenous antibiotics, and sometimes surgery. The aim of this study was to develop a mouse model of CA-MRSA wound infection to compare the efficacy of commonly used systemic and topical antibiotics. A bioluminescent USA300 CA-MRSA strain was inoculated into full-thickness scalpel wounds on the backs of mice and digital photography/image analysis and in vivo bioluminescence imaging were used to measure wound healing and the bacterial burden. Subcutaneous vancomycin, daptomycin, and linezolid similarly reduced the lesion sizes and bacterial burden. Oral linezolid, clindamycin, and doxycycline all decreased the lesion sizes and bacterial burden. Oral trimethoprim-sulfamethoxazole decreased the bacterial burden but did not decrease the lesion size. Topical mupirocin and retapamulin ointments both reduced the bacterial burden. However, the petrolatum vehicle ointment for retapamulin, but not the polyethylene glycol vehicle ointment for mupirocin, promoted wound healing and initially increased the bacterial burden. Finally, in type 2 diabetic mice, subcutaneous linezolid and daptomycin had the most rapid therapeutic effect compared with vancomycin. Taken together, this mouse model of CA-MRSA wound infection, which utilizes in vivo bioluminescence imaging to monitor the bacterial burden, represents an alternative method to evaluate the preclinical in vivo efficacy of systemic and topical antimicrobial agents.

GraS signaling in Staphylococcus aureus is regulated by a single D35 residue in the extracellular loop.

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.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-ß-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care ß-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to ß-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-ß-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most ß-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to ß-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-ß-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using ß-lactams.