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.

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.

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.

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.

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.

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.

Genetic interruption of target genes for investigation of virulence factors.

Recently, more emphasis has been given to understand molecular genetics and the contribution of a gene in the disease process. In fact, increased understanding of bacterial pathogenesis and intracellular communication has revealed many potential strategies for development of novel agents to treat bacterial infection. Therefore, to study the function and the involvement of a particular gene in pathogenesis, the inactivation or interruption is very important. In this section, various methods leading to inactivation of the gene in Staphylococcus aureus will be discussed.

Phenotypic characterization of sarR mutant in Staphylococcus aureus.

Multiple factors of Staphylococcus aureus are involved in infection. Expression of these factors is controlled by multiple regulatory systems such as, the Sar family of transcriptional regulators. The staphylococcal specific Sar family of proteins are involved in expression of numerous target genes involving virulence, autolysis, biofilm formation, antibiotic resistance, oxidative stresses, and metabolic processes. Genetic and biochemical characterization of several sar family genes have been studied. However, less is known about the phenotypic properties of the sar family mutants, except sarA mutant in S. aureus. In this report, various studies such as phenotype microarray, autolytic, hemolytic, protease and DNase assays were performed to study the phenotypic properties of sarR mutant, a member of the sar family mutants. Phenotypic microarray for growth kinetic analysis identified eight substances (e.g., chlorhexidine, ceslodin, 3,5-dinitrobenzene, plumbagin, minocycline, dipeptide Arg-Ser, phenylarsine oxide and piperacillin), whose mode of actions were more specific towards cell wall or membrane. These findings were confirmed by various independent growth study experiments. Overall, the results from various phenotypic assays such as growth kinetics, autolysis, protease and DNase suggest that a sarR mutant strain is more sensitive to autolytic activities compared to the wild type, while less sensitive with respect to a sarA mutant strain.

Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles.

The antibacterial properties of zinc oxide nanoparticles were investigated using both gram-positive and gram-negative microorganisms. These studies demonstrate that ZnO nanoparticles have a wide range of antibacterial activities toward various microorganisms that are commonly found in environmental settings. The antibacterial activity of the ZnO nanoparticles was inversely proportional to the size of the nanoparticles in S. aureus. Surprisingly, the antibacterial activity did not require specific UV activation using artificial lamps, rather activation was achieved under ambient lighting conditions. Northern analyses of various reactive oxygen species (ROS) specific genes and confocal microscopy suggest that the antibacterial activity of ZnO nanoparticles might involve both the production of reactive oxygen species and the accumulation of nanoparticles in the cytoplasm or on the outer membranes. Overall, the experimental results suggest that ZnO nanoparticles could be developed as antibacterial agents against a wide range of microorganisms to control and prevent the spreading and persistence of bacterial infections.

Control of thioredoxin reductase gene (trxB) transcription by SarA in Staphylococcus aureus.

Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sarA family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sarA regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus.

Transcriptional variation of regulatory and virulence genes due to different media in Staphylococcus aureus.

The pathogenesis of staphylococcal infections is a multifactorial process that depends on expression of different virulence factors. Expression of these factors is controlled by multiple regulatory systems in conjunction with environmental signals. Most of the genetic studies in Staphylococcus aureus have been performed using different growth media, therefore, we examined the effects of different growth media on transcription of the selective target (e.g., hla, hlb, spa, sspA) and regulatory (e.g., agr, sarA family) genes. The results from this study suggest that different growth media have substantial effect on transcription of various genes being analyzed. Interestingly, when compared with the wild-type, the isogenic sarA mutant showed a media-dependent distinct regulatory effect on expression of the target genes.

Expression of the sarA family of genes in different strains of Staphylococcus aureus.

Expression of genes involved in the pathogenesis of Staphylococcus aureus is controlled by global regulatory loci, including two-component regulatory systems and transcriptional regulators. The staphylococcal-specific SarA family of transcription regulators control large numbers of target genes involved in virulence, autolysis, biofilm formation, stress responses and metabolic processes, and are recognized as potential therapeutic targets. Expression of some of these important regulators has been examined, mostly in laboratory strains, while the pattern of expression of these genes in other strains, especially clinical isolates, is largely unknown. In this report, a comparative analysis of 10 sarA-family genes was conducted in six different S. aureus strains, including two laboratory (RN6390, SH1000) and four clinical (MW2, Newman, COL and UAMS-1) strains, by Northern and Western blot analyses. Transcription of most of the sarA-family genes showed a strong growth phase-dependence in all strains tested. Among these genes, no difference was observed in expression of the sarA, sarV, sarT and sarU genes, while a major difference was observed in expression of the sarX gene only in strain RN6390. Expression of mgrA, rot, sarZ, sarR and sarS was observed in all strains, but the level of expression varied from strain to strain.

Regulation of superoxide dismutase (sod) genes by SarA in Staphylococcus aureus.

The scavenging of reactive oxygen species (ROS) within cells is regulated by several interacting factors, including transcriptional regulators. Involvement of sarA family genes in the regulation of proteins involved in the scavenging of ROS is largely unknown. In this report, we show that under aerobic conditions, the levels of sodM and sodA transcription, in particular the sodM transcript, are markedly enhanced in the sarA mutant among the tested sarA family mutants. Increased levels of sod expression returned to near the parental level in a single-copy sarA complemented strain. Under microaerophilc conditions, transcription of both sodM and sodA was considerably enhanced in the sarA mutant compared to the wild-type strain. Various genotypic, phenotypic, and DNA binding studies confirmed the involvement of SarA in the regulation of sod transcripts in different strains of Staphylococcus aureus. The sodA mutant was sensitive to an oxidative stress-inducing agent, methyl viologen, but the sarA sodA double mutant was more resistant to the same stressor than the single sodA mutant. These results suggest that overexpression of SodM, which occurs in the sarA background, can rescue the methyl viologen-sensitive phenotype observed in the absence of the sodA gene. Analysis with various oxidative stress-inducing agents indicates that SarA may play a greater role in modulating oxidative stress resistance in S. aureus. This is the first report that demonstrates the direct involvement of a regulatory protein (SarA) in control of sod expression in S. aureus.

sarZ, a sarA family gene, is transcriptionally activated by MgrA and is involved in the regulation of genes encoding exoproteins in Staphylococcus aureus.

The expression of genes involved in the pathogenesis of Staphylococcus aureus is controlled by global regulatory loci, including two-component regulatory systems and transcriptional regulators (e.g., sar family genes). Most members of the SarA family have been partially characterized and shown to regulate a large numbers of target genes. Here, we describe the characterization of sarZ, a sarA paralog from S. aureus, and its regulatory relationship with other members of its family. Expression of sarZ was growth phase dependent with maximal expression in the early exponential phase of growth. Transcription of sarZ was reduced in an mgrA mutant and returned to a normal level in a complemented mgrA mutant strain, which suggests that mgrA acts as an activator of sarZ transcription. Purified MgrA protein bound to the sarZ promoter region, as determined by gel shift assays. Among the sarA family of genes analyzed, inactivation of sarZ increased sarS transcription, while it decreased agr transcription. The expression of potential target genes involved in virulence was evaluated in single and double mutants of sarZ with mgrA, sarX, and agr. Northern and zymogram analyses indicated that the sarZ gene product played a role in regulating several virulence genes, particularly those encoding exoproteins. Gel shift assays demonstrated nonspecific binding of purified SarZ protein to the promoter regions of the sarZ-regulated target genes. These results demonstrate the important role played by SarZ in controlling regulatory and virulence gene expression in S. aureus.