scholarly journals mgr, a Novel Global Regulator in Staphylococcus aureus

2003 ◽  
Vol 185 (13) ◽  
pp. 3703-3710 ◽  
Author(s):  
Thanh T. Luong ◽  
Steven W. Newell ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Transcriptional Level ◽  
Virulence Determinants ◽  
Alpha Toxin ◽  
Extracellular Protein Production

ABSTRACT The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

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 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.

Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents

Parasitology ◽  
1984 ◽  
Vol 88 (1) ◽  
pp. 27-36 ◽  
Author(s):  
R. J. Howard ◽  
J. W. Barnwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Plasmodium Knowlesi ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Ionic Detergents ◽  
Class A ◽  
Anionic Detergents ◽  
Triton X

SUMMARYPlasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200000 and 180000 were detected only after extraction with deoxycholate or SDS.

scholarly journals Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and protein A in pathogenesis.

1994 ◽  
Vol 62 (6) ◽  
pp. 2478-2482 ◽  
Author(s):  
M C Callegan ◽  
L S Engel ◽  
J M Hill ◽  
R J O'Callaghan
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Alpha Toxin

scholarly journals Prevalence of Methicillin Resistant and Virulence Determinants in Clinical Isolates of Staphylococcus aureus

2018 ◽  
Vol 10 (1) ◽  
pp. 108-115
Author(s):  
Manjunath Chavadi ◽  
Rahul Narasanna ◽  
Ashajyothi Chavan ◽  
Ajay Kumar Oli ◽  
Chandrakanth Kelmani. R
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Morphology ◽  
Methicillin Resistance ◽  
Protein A ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Virulence Determinants ◽  
Methicillin Resistant ◽  
Alternative Medicines ◽  
Mrsa Strains

Introduction:Methicillin-resistantStaphylococcus aureus(MRSA) is the major threat that is a result of the uncontrolled use of antibiotics causing a huge loss in health, so understanding their prevalence is necessary as a public health measure.Objective:The aim of this study was to determine the prevalence of methicillin-resistant MRSA and virulence determinant among associatedS. aureusfrom the clinical samples obtained from various hospital and health care centers of the Gulbarga region in India.Materials and Methods:All the collected samples were subjected for the screening ofS. aureusand were further characterized by conventional and molecular methods including their antibiotic profiling. Further, the response of methicillin antibiotic on cell morphology was studied using scanning electron microscopy.Results:A total 126S. aureuswas isolated from the clinical samples which showed, 100% resistant to penicillin, 55.5% to oxacillin, 75.3% to ampicillin, 70.6% to streptomycin, 66.6% to gentamicin, 8.7% to vancomycin and 6.3% to teicoplanin. The selected MRSA strains were found to possessmecA(gene coding for penicillin-binding protein 2A) andfemA(factor essential for methicillin resistance)genetic determinants in their genome with virulence determinants such as Coagulase (coa) and the X region of the protein A (spa)gene. Further, the methicillin response in resistantS. aureusshowed to be enlarged and malformed on cell morphology.Conclusion:The molecular typing of clinical isolates ofS. aureusin this study was highly virulent and also resistant to methicillin; this will assist health professionals to control, exploration of alternative medicines and new approaches to combat Staphylococcal infections more efficiently by using targeted therapy.

scholarly journals Protein Engineering of Wzc To Generate New Emulsan Analogs

2007 ◽  
Vol 73 (12) ◽  
pp. 4020-4028 ◽  
Author(s):  
Hanna Dams-Kozlowska ◽  
David L. Kaplan
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Genetic Manipulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Point Mutations ◽  
Biological Properties ◽  
Feeding Strategies ◽  
Exogenous Fatty Acid

ABSTRACT Acinetobacter venetianus Rag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modified wzc variants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant, exhibiting decreased adherence to polystyrene, and inhibited biofilm formation. The results described in this report show the functional effect of Wzc modification on the molecular weight of a high-molecular-weight polysaccharide. Moreover, in the present study we developed a genetic system to control polymerization of apoemulsan. The use of selective exogenous fatty acid feeding strategies, as well as genetic manipulation of sugar backbone chain length, is a promising new approach for bioengineering emulsan analogs.

scholarly journals Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 599-606 ◽  
Author(s):  
MJ Telen ◽  
TJ Palker ◽  
BF Haynes
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Two Dimensions ◽  
Antigen Activity

Abstract We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.

cAMP increases synthesis of surfactant-associated protein A by perfused rat lung

1991 ◽  
Vol 260 (4) ◽  
pp. L226-L233 ◽  
Author(s):  
A. B. Fisher ◽  
I. Arad ◽  
C. Dodia ◽  
A. Chander ◽  
S. I. Feinstein
Keyword(s):  
Protein Fraction ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Synthetic Medium ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Lag Phase

Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8-BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27–36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant-associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A.

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