scholarly journals Regulation of Rot Expression in Staphylococcus aureus

2007 ◽  
Vol 190 (2) ◽  
pp. 546-554 ◽  
Author(s):  
Hsin-Yeh Hsieh ◽  
Ching Wen Tseng ◽  
George C. Stewart
Keyword(s):  
Staphylococcus Aureus ◽  
Affinity Purification ◽  
Protein A ◽  
Protein Sequencing ◽  
Dependent Manner ◽  
Accessory Gene ◽  
Mobility Shift ◽  
Virulence Gene Expression ◽  
Promoter Fragment ◽  
Repressive Effect

ABSTRACT Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in Staphylococcus aureus. The function of Rot, but not the transcription of rot, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (σB) has a repressive effect on rot expression during the postexponential phase of growth. The transcriptional profiles of Rot in σB-positive and σB-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of rot expression was observed during the stationary phase of growth, and this upregulation occurred in a σB-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the rot promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of rot expression was also observed in sarS-negative strains. Affinity purification of proteins binding to the rot promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the rot promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the rot mRNA but not the inhibition of rot transcription.

scholarly journals N-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in Staphylococcus aureus

2006 ◽  
Vol 74 (2) ◽  
pp. 910-919 ◽  
Author(s):  
Saara Qazi ◽  
Barry Middleton ◽  
Siti Hanna Muharram ◽  
Alan Cockayne ◽  
Philip Hill ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Protein A ◽  
Light Output ◽  
Virulence Gene ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Virulence Gene Expression ◽  
Growth Inhibitory

ABSTRACT Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 μM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.

scholarly journals Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

2006 ◽  
Vol 50 (4) ◽  
pp. 1183-1194 ◽  
Author(s):  
Kati Seidl ◽  
Martin Stucki ◽  
Martin Ruegg ◽  
Christiane Goerke ◽  
Christiane Wolz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Virulence Gene ◽  
Exponential Growth Phase ◽  
Virulence Determinants ◽  
Resistance Level ◽  
Virulence Gene Expression ◽  
Oxacillin Resistance

ABSTRACT Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

scholarly journals CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Marat R. Sadykov ◽  
Ian H. Windham ◽  
Todd J. Widhelm ◽  
Vijaya Kumar Yajjala ◽  
Sean M. Watson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Carbon Metabolism ◽  
Protein A ◽  
Transcriptional Regulators ◽  
Extracellular Dna ◽  
Regulatory Control ◽  
Pcr Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Biofilm Development

ABSTRACT The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of PcidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus. The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

scholarly journals Proteomic and Transcriptomic Profiling of Staphylococcus aureus Surface LPXTG-proteins: Correlation with agr Genotypes and Adherence Phenotypes

2012 ◽  
Vol 11 (11) ◽  
pp. 1123-1139 ◽  
Author(s):  
Mathilde Ythier ◽  
Grégory Resch ◽  
Patrice Waridel ◽  
Alexandre Panchaud ◽  
Aurélie Gfeller ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Regulatory Networks ◽  
Time Course ◽  
Methicillin Resistance ◽  
Binding Protein ◽  
Protein A ◽  
Surface Proteins ◽  
Protein Domain ◽  
Accessory Gene

Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.

scholarly journals Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

2008 ◽  
Vol 191 (3) ◽  
pp. 832-843 ◽  
Author(s):  
Nekane Merino ◽  
Alejandro Toledo-Arana ◽  
Marta Vergara-Irigaray ◽  
Jaione Valle ◽  
Cristina Solano ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Protein A ◽  
Surface Proteins ◽  
Growth Media ◽  
Dependent Manner ◽  
Chronic Infections ◽  
The Matrix ◽  
Implanted Medical Devices ◽  
Biofilm Development

ABSTRACT The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

scholarly journals MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

2006 ◽  
Vol 50 (4) ◽  
pp. 1276-1281 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Carmen E. DeMarco ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Multidrug Efflux ◽  
Recognition Sequence ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Repressive Effect ◽  
Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

scholarly journals Staphylococcus aureus CodY Negatively Regulates Virulence Gene Expression

2007 ◽  
Vol 190 (7) ◽  
pp. 2257-2265 ◽  
Author(s):  
Charlotte D. Majerczyk ◽  
Marat R. Sadykov ◽  
Thanh T. Luong ◽  
Chia Lee ◽  
Greg A. Somerville ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Regulatory Protein ◽  
Virulence Gene ◽  
Culture Fluid ◽  
Accessory Gene ◽  
Virulence Gene Expression ◽  
Gram Positive Bacteria ◽  
Accessory Gene Regulator ◽  
Branched Chain

ABSTRACT CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus.

scholarly journals Subinhibitory Concentrations of Linezolid Reduce Staphylococcus aureus Virulence Factor Expression

2004 ◽  
Vol 48 (2) ◽  
pp. 546-555 ◽  
Author(s):  
Katussevani Bernardo ◽  
Norbert Pakulat ◽  
Silke Fleer ◽  
Annabelle Schnaith ◽  
Olaf Utermöhlen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Elongation Factor ◽  
Dependent Manner ◽  
Triacylglycerol Lipase ◽  
Subinhibitory Concentrations ◽  
Dose Dependent

ABSTRACT The influence of the antibiotic linezolid on the secretion of exotoxins by Staphylococcus aureus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot analysis. S. aureus suspensions were treated with grading subinhibitory concentrations of linezolid (12.5, 25, 50, and 90% of MIC) at different stages of bacterial growth (i.e., an optical density at 540 nm [OD540] of 0.05 or 0.8). When added to S. aureus cultures at an OD540 of 0.05, linezolid reduced in a dose-dependent manner the secretion of specific virulence factors, including staphylococcal enterotoxin A (SEA) and SEB, bifunctional autolysin, autolysin, protein A, and alpha- and beta-hemolysins. In contrast, other presumably nontoxic exoproteins remained unchanged or even accumulated in supernatants in the presence of linezolid at a 90% MIC. Similarily, when added at OD540 of 0.8, that is, after quorum sensing, linezolid reduced the release of virulence factors, whereas the relative abundance of nontoxic exoproteins such as triacylglycerol lipase, glycerol ester hydrolase, DnaK, or translation elongation factor EF-Tu was found to be increased. Consistently, linezolid reduced in a dose-dependent manner the tumor necrosis factor-inducing activity secreted by S. aureus into the culture supernatants. The results of our study suggest that the expression of virulence factors in S. aureus is especially sensitive to the inhibition of protein synthesis by linezolid, which should be an advantage in the treatment of infections with toxin-producing S. aureus.

scholarly journals A Novel Aza-Derivative Inhibits agr Quorum Sensing Signaling and Synergizes Methicillin-Resistant Staphylococcus aureus to Clindamycin

2021 ◽  
Vol 12 ◽  
Author(s):  
Giulia Bernabè ◽  
Matteo Dal Pra ◽  
Vittoria Ronca ◽  
Anthony Pauletto ◽  
Giovanni Marzaro ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Response Regulator ◽  
Accessory Gene ◽  
Mobility Shift ◽  
Signaling Mechanism ◽  
Methicillin Resistant ◽  
Qrt Pcr ◽  
Mobility Shift Assay

Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors’ expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.

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