scholarly journals Transcriptional Profiling of Target of RNAIII-Activating Protein, a Master Regulator of Staphylococcal Virulence

2005 ◽  
Vol 73 (10) ◽  
pp. 6220-6228 ◽  
Author(s):  
Moshe Korem ◽  
Yael Gov ◽  
Madanahally D. Kiran ◽  
Naomi Balaban
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Transcriptional Profiling ◽  
Protein A ◽  
Cell Communication ◽  
Global Regulator ◽  
Positive Bacterium ◽  
Master Regulator ◽  
Expression Of Genes ◽  
Histidine Phosphorylation

ABSTRACT Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.

scholarly journals Phosphorylation of MgrA and Its Effect on Expression of the NorA and NorB Efflux Pumps of Staphylococcus aureus

2010 ◽  
Vol 192 (10) ◽  
pp. 2525-2534 ◽  
Author(s):  
Que Chi Truong-Bolduc ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Multidrug Resistance ◽  
Virulence Factors ◽  
Double Mutant ◽  
Efflux Pumps ◽  
Efflux Transporters ◽  
Global Regulator ◽  
Binding Assays ◽  
Genes Encoding ◽  
Gel Shift

ABSTRACT MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrAS110A-S113A bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

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 The Staphylococcus aureus GGDEF Domain-Containing Protein, GdpS, Influences Protein A Gene Expression in a Cyclic Diguanylic Acid-Independent Manner

2009 ◽  
Vol 77 (7) ◽  
pp. 2849-2856 ◽  
Author(s):  
Fei Shang ◽  
Ting Xue ◽  
Haipeng Sun ◽  
Lei Xing ◽  
Shuo Zhang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Protein A ◽  
Surface Protein ◽  
Transcript Level ◽  
Cysteine Proteases ◽  
Independent Manner ◽  
Wide Range ◽  
Ggdef Domain ◽  
Major Surface

ABSTRACT Staphylococcus aureus is an important human pathogen that is the principal cause of a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of the organism as a pathogen and its ability to cause such a wide range of infections are due to its extensive virulence factors. In this study, we identified the role of the only GGDEF domain protein (GdpS [GGDEF domain protein from Staphylococcus]) in the virulence of S. aureus NCTC8325. Inactivation of gdpS results in an alteration in the production of a range of virulence factors, such as serine and cysteine proteases, fibrinogen-binding proteins, and, specifically, protein A (Spa), a major surface protein of S. aureus. The transcript level of spa decreases eightfold in the gdpS mutant compared with the parental NCTC8325 strain. Furthermore, the transcript level of sarS, which encodes a direct positive regulator of spa, also decreases in the gdpS mutant compared with the wild type, while the transcript levels of agr, sarA, sarT, and rot display no apparent changes in the gdpS mutant, suggesting that GdpS affects the expression of spa through interaction with SarS by unknown mechanisms. Furthermore, the complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C-terminal GGDEF domain with conserved GGDEF, suggesting that GdpS functions in S. aureus by an unknown mechanism independent of 3′,5′-cyclic diguanylic acid signaling.

scholarly journals Transcriptional Profiling of XdrA, a New Regulator of spa Transcription in Staphylococcus aureus

2010 ◽  
Vol 192 (19) ◽  
pp. 5151-5164 ◽  
Author(s):  
N. McCallum ◽  
J. Hinds ◽  
M. Ender ◽  
B. Berger-Bächi ◽  
P. Stutzmann Meier
Keyword(s):  
Staphylococcus Aureus ◽  
Reporter Gene ◽  
Transcriptional Profiling ◽  
Protein A ◽  
Dna Binding Protein ◽  
Infection Process ◽  
Gene Fusions ◽  
Temporal Expression ◽  
Nested Deletions ◽  
Complex Regulatory Network

ABSTRACT Transcription of spa, encoding the virulence factor protein A in Staphylococcus aureus, is tightly controlled by a complex regulatory network, ensuring its temporal expression over growth and at appropriate stages of the infection process. Transcriptomic profiling of XdrA, a DNA-binding protein that is conserved in all S. aureus genomes and shares similarity with the XRE family of helix-turn-helix, antitoxin-like proteins, revealed it to be a previously unidentified activator of spa transcription. To assess how XdrA fits into the complex web of spa regulation, a series of regulatory mutants were constructed; consisting of single, double, triple, and quadruple mutants lacking XdrA and/or the three key regulators previously shown to influence spa transcription directly (SarS, SarA, and RNAIII). A series of lacZ reporter gene fusions containing nested deletions of the spa promoter identified regions influenced by XdrA and the other three regulators. XdrA had almost as strong an activating effect on spa as SarS and acted on the same spa operator regions as SarS, or closely overlapping regions. All data from microarrays, Northern and Western blot analyses, and reporter gene fusion experiments indicated that XdrA is a major activator of spa expression that appears to act directly on the spa promoter and not through previously characterized regulators.

scholarly journals Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis in Rio de Janeiro

2009 ◽  
Vol 29 (5) ◽  
pp. 369-374 ◽  
Author(s):  
Shana M.O. Coelho ◽  
Elina Reinoso ◽  
Ingrid A. Pereira ◽  
Lidiane C. Soares ◽  
Mirta Demo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Protein A ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Resistance Pattern ◽  
Igg Binding ◽  
Amplicon Size ◽  
Resistance To Penicillin ◽  
Single Amplicon

The study was conducted to characterize pheno-genotypically the virulence factors and resistance pattern of Staphylococcus aureus isolates from milk samples of cows with subclinical mastitis. All hemolytic isolates presented beta-hemolysin, and 38% of the non-hemolytic isolates were able to express hemolysins in the presence of a beta-hemolytic strain. The amplification of the coa-gene displayed four different size polymorphisms with about 400 bp, 600 bp, 700 bp and 900 bp. The spaA gene that encodes the IgG-binding region of protein A revealed sizes of 700 bp and 900 bp. The amplification of region X from spaA yielded a single amplicon for each isolate with the prevalent amplicon size being of 180 bp. Amplification of sae gene yielded an amplicon size of 920 bp in 71% of the isolates. Antibiotic resistance pattern revealed that 42% S. aureus were susceptible to all antimicrobials tested. Seven different antibiotic patterns were observed. Our results indicated that 47% and 25% of S. aureus strains exhibited resistance to penicillin and oxacillin respectively. All oxacillin-resistant isolates were mecA-positive.

scholarly journals Regulation of Exoprotein Gene Expression by the Staphylococcus aureus cvfB Gene

2007 ◽  
Vol 75 (4) ◽  
pp. 1964-1972 ◽  
Author(s):  
Yasuhiko Matsumoto ◽  
Chikara Kaito ◽  
Daisuke Morishita ◽  
Kenji Kurokawa ◽  
Kazuhisa Sekimizu
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Genetic Background ◽  
Promoter Activity ◽  
Double Mutant ◽  
Gene Deletion ◽  
Protein A ◽  
Protease Production ◽  
Gene Deletion Mutant

ABSTRACT We previously reported that the cvfB gene (SA1223) of Staphylococcus aureus is responsible for the virulence of this pathogenic bacterium. We show here that the cvfB gene regulates exoprotein gene expression. In a cvfB gene deletion mutant, hemolysin, DNase, and protease production were decreased, whereas protein A expression was increased. The amount of RNAIII, the transcript from the P3 promoter in the agr locus that regulates the expression of various virulence factors, was also reduced in the cvfB mutant. In addition, P2 and P3 promoter activity in the agr locus was decreased in the mutant. Under the genetic background of the agr-null mutation, cvfB gene disruption decreased the production levels of DNase and protease. Moreover, the cvfB and agr double mutant was less virulent than the agr mutant in silkworms. These results suggest that the cvfB gene product contributes to the expression of virulence factors and to pathogenicity via both agr-dependent and agr-independent pathways.

scholarly journals Characterization of Virulence Factor Regulation by SrrAB, a Two-Component System in Staphylococcus aureus

2004 ◽  
Vol 186 (8) ◽  
pp. 2430-2438 ◽  
Author(s):  
Alexa A. Pragman ◽  
Jeremy M. Yarwood ◽  
Timothy J. Tripp ◽  
Patrick M. Schlievert
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Transcriptional Start Site ◽  
Protein A ◽  
Rabbit Model ◽  
Low Oxygen ◽  
Two Component System ◽  
Component System ◽  
Two Component

ABSTRACT Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

scholarly journals Identification and characterization of msa (SA1233), a gene involved in expression of SarA and several virulence factors in Staphylococcus aureus

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2559-2572 ◽  
Author(s):  
Karthik Sambanthamoorthy ◽  
Mark S. Smeltzer ◽  
Mohamed O. Elasri
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Protein A ◽  
Laboratory Strain ◽  
Reporter System ◽  
Accessory Gene ◽  
Accessory Gene Regulator ◽  
Genes Encoding ◽  
Membrane Spanning ◽  
The Impact

The staphylococcal accessory regulator (sarA) plays a central role in the regulation of virulence in Staphylococcus aureus. To date, studies involving sarA have focused on its activity as a global regulator that modulates transcription of a wide variety of genes (>100) and its role in virulence. However, there is also evidence to suggest the existence of accessory elements that modulate SarA production and/or function. A reporter system was developed to identify such elements, and a new gene, msa (SA1233), mutation of which results in reduced expression of SarA, was identified and characterized. Additionally, it was shown that mutation of msa resulted in altered transcription of the accessory gene regulator (agr) and the genes encoding several virulence factors including alpha toxin (hla) and protein A (spa). However, the impact of mutating msa was different in the laboratory strain RN6390 and the clinical isolate UAMS-1. For instance, mutation of msa caused a decrease in spa and hla transcription in RN6390 but had a different effect in UAMS-1. The strain-dependent effects of the msa mutation were similar to those observed previously, which suggests that msa may modulate the production of specific virulence factors through its impact on sarA. Interestingly, sequence analysis of Msa suggests that it is a putative membrane protein with three membrane-spanning regions, indicating that Msa might interact with the environment. The findings show that msa is involved in the expression of SarA and several virulence factors.

scholarly journals MsrR, a Putative Cell Envelope-Associated Element Involved in Staphylococcus aureus sarA Attenuation

2003 ◽  
Vol 47 (8) ◽  
pp. 2558-2564 ◽  
Author(s):  
Jutta Rossi ◽  
Markus Bischoff ◽  
Akihito Wada ◽  
Brigitte Berger-Bächi
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Regulatory Network ◽  
Cell Envelope ◽  
Protein A ◽  
Virulence Gene ◽  
Exponential Growth Phase ◽  
Alpha Toxin ◽  
Altered Expression ◽  
Virulence Gene Expression

ABSTRACT A novel membrane-associated protein, MsrR, was identified in Staphylococcus aureus which affects resistance to methicillin and teicoplanin, as well as the synthesis of virulence factors. MsrR belongs to the LytR-CpsA-Psr family of cell envelope-related transcriptional attenuators and was shown to be inducible by cell wall-active agents, such as β-lactams, glycopeptides, and lysostaphin. The expression of msrR peaked in the early exponential growth phase and decreased sharply thereafter. msrR mutants showed increased sarA transcription and an earlier and higher expression of RNAIII, resulting in altered expression of virulence factors such as alpha-toxin and protein A. These observations suggest that MsrR is a new component involved in sarA attenuation and the regulatory network controlling virulence gene expression in S. aureus.

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