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.

Gp05, a Prophage-Encoded Virulence Factor, Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a serious public health threat. We recently demonstrated that the presence of a novel prophage ϕSA169 was associated with vancomycin (VAN) treatment failure in experimental MRSA endocarditis. In this study, we assessed the role of a ϕSA169 gene, ϕ80α_gp05 (gp05), in VAN-persistent outcome using gp05 isogenic MRSA strain sets. Of note, Gp05 significantly influences the intersection of MRSA virulence factors, host immune responses, and antibiotic treatment efficacy, including the following: (i) activity of the significant energy-yielding metabolic pathway (e.g., tricarboxylic acid cycle); (ii) carotenoid pigment production; (iii) (p)ppGpp (guanosine tetra- and pentaphosphate) production, which activates the stringent response and subsequent downstream functional factors (e.g., phenol-soluble modulins and polymorphonuclear neutrophil bactericidal activity); and (iv) persistence to VAN treatment in an experimental infective endocarditis model. These data suggest that Gp05 is a significant virulence factor which contributes to the persistent outcomes in MRSA endovascular infection by multiple pathways. IMPORTANCE Persistent endovascular infections are often caused by MRSA strains that are susceptible to anti-MRSA antibiotics in vitro by CLSI breakpoints. Thus, the persistent outcome represents a unique variant of traditional antibiotic resistance mechanisms and a significant therapeutic challenge. Prophage, a critical mobile genetic element carried by most MRSA isolates, provides their bacterial host with metabolic advantages and resistance mechanisms. However, how prophage-encoded virulence factors interact with the host defense system and antibiotics, driving the persistent outcome, is not well known. In the current study, we demonstrated that a novel prophage gene, gp05, significantly impacts tricarboxylic acid cycle activity, stringent response, and pigmentation, as well as vancomycin treatment outcome in an experimental endocarditis model using isogenic gp05 overexpression and chromosomal deletion mutant MRSA strain sets. The findings significantly advance our understanding of the role of Gp05 in persistent MRSA endovascular infection and provide a potential target for development of novel drugs against these life-threatening infections.

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.

Influence of Sodium Bicarbonate on Wall Teichoic Acid Synthesis and ß-Lactam Sensitization in NaHCO3-Responsive and Nonresponsive Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) strains pose major treatment challenges due to their innate resistance to most ß-lactams under standard in vitro antimicrobial susceptibility testing conditions. A novel phenotype among MRSA, termed "NaHCO3 responsiveness," where certain strains display increased susceptibility to ß-lactams in the presence of NaHCO3, has been identified among a relatively large proportion of MRSA isolates. One underlying mechanism of NaHCO3 responsiveness appears to be related to decreased expression and altered functionality of several genes and proteins involved in cell wall synthesis and maturation. Here, we studied the impact of NaHCO3 on wall teichoic acid (WTA) synthesis, a process intimately linked to peptidoglycan (PG) synthesis and functionality, in NaHCO3-responsive versus -nonresponsive MRSA isolates. NaHCO3 sensitized responsive MRSA strains to cefuroxime, a specific penicillin-binding protein 2 (PBP2)-inhibitory ß-lactam known to synergize with early WTA synthesis inhibitors (e.g., ticlopidine). Combining cefuroxime with ticlopidine with or without NaHCO3 suggested that these latter two agents target the same step in WTA synthesis. Further, NaHCO3 decreased the abundance and molecular weight of WTA only in responsive strains. Additionally, NaHCO3 stimulated increased autolysis and aberrant cell division in responsive strains, two phenotypes associated with disruption of WTA synthesis. Of note, studies of key genes involved in the WTA biosynthetic pathway (e.g., tarO, tarG, dltA, and fmtA) indicated that the inhibitory impact of NaHCO3 on WTA biosynthesis in responsive strains likely occurred posttranslationally. IMPORTANCE MRSA is generally viewed as resistant to standard ß-lactam antibiotics. However, a NaHCO3-responsive phenotype is observed in a substantial proportion of clinical MRSA strains in vitro, i.e., isolates which demonstrate enhanced susceptibility to standard ß-lactam antibiotics (e.g., oxacillin) in the presence of NaHCO3. This phenotype correlates with increased MRSA clearance in vivo by standard ß-lactam antibiotics, suggesting that patients with infections caused by such MRSA strains might be amenable to treatment with ß-lactams. The mechanism(s) behind this phenotype is not fully understood but appears to involve mecA-PBP2a production and maturation axes. Our study adds significantly to this body of knowledge in terms of additional mechanistic targets of NaHCO3 in selected MRSA strains. This investigation demonstrates that NaHCO3 has direct impacts on S. aureus wall teichoic acid biosynthesis in NaHCO3-responsive MRSA. These findings provide an additional target for new agents being designed to synergistically kill MRSA using ß-lactam antibiotics.

Identification of 5-(Aryl/Heteroaryl)amino-4-quinolones as Potent Membrane-Disrupting Agents to Combat Antibiotic-Resistant Gram-Positive Bacteria.

Nosocomial infections caused by resistant Gram-positive organisms are on the rise, presumably due to a combination of factors including prolonged hospital exposure, increased use of invasive procedures, and pervasive antibiotic therapy. Although antibiotic stewardship and infection control measures are helpful, newer agents against multidrug-resistant (MDR) Gram-positive bacteria are urgently needed. Here, we describe our efforts that led to the identification of 5-amino-4-quinolone 111 with exceptionally potent Gram-positive activity with minimum inhibitory concentrations (MICs) ≤0.06 µg/mL against numerous clinical isolates. Preliminary mechanism of action and resistance studies demonstrate that the 5-amino-4-quinolones are bacteriostatic, do not select for resistance, and selectively disrupt bacterial membranes. While the precise molecular mechanism has not been elucidated, the lead compound is nontoxic displaying a therapeutic index greater than 500, is devoid of hemolytic activity, and has attractive physicochemical properties (clog P = 3.8, molecular weight (MW) = 441) that warrant further investigation of this promising antibacterial scaffold for the treatment of Gram-positive infections.

Teg58, a small regulatory RNA, is involved in regulating arginine biosynthesis and biofilm formation in Staphylococcus aureus.

Staphylococcus aureus adapts to different environments by sensing and responding to diverse environmental cues. The responses are coordinately regulated by regulatory proteins, and small regulatory RNAs at the transcriptional and translational levels. Here, we characterized teg58, a SarA repressed sRNA, using ChIP-Seq and RNA-Seq analysis of a sarA mutant. Phenotypic and genetic analyses indicated that inactivation of teg58 led to reduced biofilm formation in a process that is independent of SarA, agr, PIA, and PSMs. RNA-Seq analysis of teg58 mutant revealed up-regulation of arginine biosynthesis genes (i.e., argGH) as well as the ability of the mutant to grow in a chemical defined medium (CDM) lacking L-arginine. Exogenous L-arginine or endogenous induction of argGH led to decreased biofilm formation in parental strains. Further analysis in vitro and in vivo demonstrated that the specific interaction between teg58 and the argGH occurred at the post-transcriptional level to repress arginine synthesis. Biochemical and genetic analyses of various arginine catabolic pathway genes demonstrated that the catabolic pathway did not play a significant role in reduced biofilm formation in the teg58 mutant. Overall, results suggest that teg58 is a regulatory sRNA that plays an important role in modulating arginine biosynthesis and biofilm formation in S. aureus.

The thematic role of extracellular loop of VraG in activation of the membrane sensor GraS in a cystic fibrosis MRSA strain differs in nuance from the CA-MRSA strain JE2.

Patients with cystic fibrosis (CF) often suffer recurrent bronchial bacterial infections that lead to deterioration of lung function over time. The infections in CF patients are often due to S. aureus and P. aeruginosa that colonize the airways. Significantly, methicillin-resistant S. aureus (MRSA) makes it challenging for treatment in CF patients due to its feature of multiple antibiotic resistance. In bronchial airways, cationic antimicrobial peptides are often present in mucosa cells, neutrophils, and macrophages that interfere with bacterial proliferation. The major mechanism for resistance to the bactericidal activity of cationic peptides in S. aureus is mediated by the GraRS two-component system that activates expression of MprF and DltABCD to increase surface positive charge to repel interactions with cationic peptides. We recently found that VraG, a membrane permease component of the VraFG efflux pumps, harbors a long 200-residue extracellular loop (EL) that utilizes K380 to interact with the negatively charged 9-residue extracellular loop of the membrane sensor GraS to control mprF expression in a community-acquired MRSA strain JE2. In this study, we extended this observation to a CF MRSA strain CF32A1 where we affirmed that the EL loop of VraG controls GraS-mediated signal transduction; however, in contrast to community acquired MRSA strain JE2, the CF MRSA strain CF32A1 requires both K380 and K388 in the EL of VraG to properly modulate signal transduction mediated by GraS. This effect was not attributable to the several single nucleotide polymorphisms that exist between VraG and GraS in the two MRSA strains.

CF-Seq, an accessible web application for rapid re-analysis of cystic fibrosis pathogen RNA sequencing studies.

Researchers studying cystic fibrosis (CF) pathogens have produced numerous RNA-seq datasets which are available in the gene expression omnibus (GEO). Although these studies are publicly available, substantial computational expertise and manual effort are required to compare similar studies, visualize gene expression patterns within studies, and use published data to generate new experimental hypotheses. Furthermore, it is difficult to filter available studies by domain-relevant attributes such as strain, treatment, or media, or for a researcher to assess how a specific gene responds to various experimental conditions across studies. To reduce these barriers to data re-analysis, we have developed an R Shiny application called CF-Seq, which works with a compendium of 128 studies and 1,322 individual samples from 13 clinically relevant CF pathogens. The application allows users to filter studies by experimental factors and to view complex differential gene expression analyses at the click of a button. Here we present a series of use cases that demonstrate the application is a useful and efficient tool for new hypothesis generation. (CF-Seq: http://scangeo.dartmouth.edu/CFSeq/ ).

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 NaHCO3-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most ß-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed 'NaHCO3-responsiveness', is a property whereby strains are susceptible in vitro to many ß-lactams in the presence of NaHCO3. Specific mecA genotypes, repression of mecA/PBP2a expression and perturbed maturation of PBP2a by NaHCO3 have all been associated with this phenotype. The aim of this study was to define the relationship between specific mecA genotypes and PBP2a substitutions, on the one hand, with NaHCO3-responsiveness in vitro. Mutations were made in the mecA ribosomal binding site (RBS -7) and at amino acid position 246 of its coding region in parental strains MW2 (NaHCO3-responsive) and C36 (NaHCO3- nonresponsive) to generate 'swap' variants, each harboring the other's mecA-RBS/coding region genotypes. Successful swaps were confirmed by both sequencing, as well as predicted swap of in vitro penicillin-clavulanate susceptibility phenotypes. MW2 swap variants harboring the nonresponsive mecA genotypes became NaHCO3-nonresponsive (resistant to the ß-lactam, oxacillin [OXA]), in the presence of NaHCO3. Moreover, these swap variants had lost NaHCO3-mediated repression of mecA/PBP2a expression. In contrast, C36 swap variants harboring the NaHCO3-responsive mecA genotypes remained NaHCO3-nonresponsive phenotypically, and still exhibited nonrepressible mecA/PBP2a expression. These data demonstrate that in addition to the mecA genotype, NaHCO3-responsiveness may also depend on strain-specific genetic backgrounds.

Impacts of NaHCO3 on ß-Lactam Binding to PBP2a Protein Variants Associated with the NaHCO3-Responsive versus NaHCO3-Non-Responsive Phenotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to ß-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many ß-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed "NaHCO3-responsiveness". This phenotype features ß-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on ß-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-ß-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface ß-lactam binding, as well as ultimate PBP2a binding of ß-lactams.

New Mechanistic Insights into Purine Biosynthesis with Second Messenger c-di-AMP in Relation to Biofilm-Related Persistent Methicillin-Resistant Staphylococcus aureus Infections.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus infections. We have recently demonstrated that purine biosynthesis plays an important role in such persistent infections. Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the purine biosynthesis pathway and c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower ADP, ATP, and c-di-AMP levels; (ii) less biofilm formation with decreased extracellular DNA (eDNA) levels and Triton X-100-induced autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase enzyme required for c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular infection model. These results reveal an important intersection between purine biosynthesis and c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular infections. This signaling pathway represents a logical therapeutic target against persistent MRSA infections. IMPORTANCE Persistent endovascular infections caused by MRSA, including vascular graft infection syndromes and infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are "susceptible" in vitro to conventional anti-MRSA antibiotics, such as VAN and daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints. Yet, these antibiotics frequently fail to eliminate these infections in vivo. Therefore, the persistent outcomes in MRSA infections represent a unique and important variant of classic "antibiotic resistance" that is only disclosed during in vivo antibiotic treatment. Given the high morbidity and mortality associated with the persistent infection, there is an urgent need to understand the specific mechanism(s) of this syndrome. In the current study, we demonstrate that a functional intersection between purine biosynthesis and the second messenger c-di-AMP plays an important role in VAN persistence in experimental MRSA endocarditis. Targeting this pathway may represent a potentially novel and effective strategy for treating these life-threatening infections.

Expanding the Staphylococcus aureus SarA Regulon to Small RNAs.

SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the sarA regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a ΔsarA mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of sprG2, a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and in silico methodologies are effective to characterize transcriptional factor (TF) regulons and functions. IMPORTANCE Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of sprG2, a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections.

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 µM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

The Staphylococcus aureus toxin-antitoxin system YefM-YoeB is associated with antibiotic tolerance and extracellular dependent biofilm formation.

The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges for treating periprosthetic joint infection. The toxin-antitoxin system, YefM-YoeB, is thought to be a regulator for antibiotic tolerance, but its physiological role is unknown. The objective of this study was to determine the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB homologs. We hypothesized the toxin-antitoxin yoeB homologs contribute to biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2 wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT strains had higher polysaccharide intercellular adhesin (PIA) production. Treatment with sodium metaperiodate increased biofilm formation in Newman WT, indicating biofilm formation may be increased under conditions of oxidative stress. DNase I treatment decreased biofilm formation in Newman WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher extracellular DNA (eDNA) content in comparison to yoeB mutants but no differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic tolerance, and virulence.

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.

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.

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.

Impact of the Novel Prophage ϕSA169 on Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections are life-threatening syndromes with few therapeutic options. The potential impact of bacteriophages on the persistent outcome has not been well studied. In this study, we investigated the role of a novel prophage (ϕSA169) in MRSA persistence by using a lysogen-free clinically resolving bacteremia (RB) isolate and comparing it to a derivative which was obtained by infecting the RB strain with ϕSA169, which has been lysogenized in a clinical persistent MRSA bacteremia (PB) isolate. Similar to the PB isolate, the ϕSA169-lysogenized RB strain exhibited well-defined in vitro and in vivo phenotypic and genotypic signatures related to the persistent outcome, including earlier activation of global regulators (i.e., sigB, sarA, agr RNAIII, and sae); higher expression of a critical purine biosynthesis gene, purF; and higher growth rates accompanied by lower ATP levels and vancomycin (VAN) susceptibility and stronger δ-hemolysin and biofilm formation versus its isogenic parental RB isolate. Notably, the contribution of ϕSA169 in persistent outcome with VAN treatment was confirmed in an experimental infective endocarditis model. Taken together, these results indicate the critical role of the prophage ϕSA169 in persistent MRSA endovascular infections. Further studies are needed to identify the mechanisms of ϕSA169 in mediating the persistence, as well as establishing the scope of impact, of this prophage in other PB strains.IMPORTANCE Bacteriophages are viruses that invade the bacterial host, disrupt bacterial metabolism, and cause the bacterium to lyse. Because of its remarkable antibacterial activity and unique advantages over antibiotics, for instance, bacteriophage is specific for one species of bacteria and resistance to phage is less common than resistance to antibiotics. Indeed, bacteriophage therapy for treating infections due to multidrug-resistant pathogens in humans has become a research hot spot. However, it is also worth considering that bacteriophages are transferable and could cotransfer host chromosomal genes, e.g., virulence and antimicrobial resistance genes, while lysogenizing and integrating into the bacterial chromosome (prophage), thus playing a role in bacterial evolution and virulence. In the current study, we identified a novel prophage, ϕSA169, from a clinical persistent MRSA bacteremia isolate, and we determined that ϕSA169 mediated well-defined in vitro and in vivo phenotypic and genotypic signatures related to the persistent outcome, which may represent a unique and important persistent mechanism(s).

The Stringent Response Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection Through the Purine Biosynthetic Pathway.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinical-therapeutic challenge. Of particular concern is antibiotic treatment failure in infections caused by MRSA that are "susceptible" to antibiotic in vitro. In the current study, we investigate specific purine biosynthetic pathways and stringent response mechanism(s) related to this life-threatening syndrome using genetic matched persistent and resolving MRSA clinical bacteremia isolates (PB and RB, respectively), and isogenic MRSA strain sets. We demonstrate that PB isolates (vs RB isolates) have significantly higher (p)ppGpp production, phenol-soluble-modulin expression, polymorphonuclear leukocyte lysis and survival, fibronectin/endothelial cell (EC) adherence, and EC damage. Importantly, an isogenic strain set, including JE2 parental, relP-mutant and relP-complemented strains, translated the above findings into significant outcome differences in an experimental endocarditis model. These observations indicate a significant regulation of purine biosynthesis on stringent response, and suggest the existence of a previously unknown adaptive genetic mechanism in persistent MRSA infection.