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.

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 Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 810-817
Author(s):  
KJ Balazovich ◽  
JE Smolen ◽  
LA Boxer
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Dose Dependent

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

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.

scholarly journals Subinhibitory Concentrations of Prim-O-Glucosylcimifugin Decrease the Expression of Alpha-Hemolysin inStaphylococcus aureus(USA300)

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Ouyang Ping ◽  
Yang Ruixue ◽  
Deng Jiaqiang ◽  
Wang Kaiyu ◽  
Fang Jing ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
A549 Cells ◽  
Opportunistic Pathogen ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Chinese Medicines ◽  
Alpha Hemolysin ◽  
Subinhibitory Concentrations ◽  
Dose Dependent

Staphylococcus aureus (S. aureus), an important opportunistic pathogen in human and animal, causes a series of diseases in the impairing of immunity of host and even then death. Alpha-hemolysin (Hla), a primary virulence factor, plays a major role in the pathogenic progress ofS. aureus, especially in pneumonia. Prim-O-glucosylcimifugin (POG), a nature chromone compound, is an active ingredient in many Chinese Medicines. In this study, POG investigated the inhibitory effect of the secretion of Hla inS. aureusstrain USA300 at the subinhibitory concentrations. The hemolysis assays and western blotting assays showed that POG can decrease the production of Hla in the USA300 growth cell cultures in a dose-dependent manner. The results of RT-PCR revealed that reduction of Hla was related to inhibit the transcription ofhlaandRNAIII. In the cells experiment, POG was proved to protect A549 cells from Hla-medicated injury. In conclusion, POG was shown the capacity of decreased the production ofS. aureusHla. POG can be developed as a candidate agent to treatS. aureusinfections against Hla.

scholarly journals Influence of Nisin-Biogel at Subinhibitory Concentrations on Virulence Expression in Staphylococcus aureus Isolates from Diabetic Foot Infections

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1501
Author(s):  
Carolina Jesus ◽  
Rui Soares ◽  
Eva Cunha ◽  
Miguel Grilo ◽  
Luís Tavares ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Diabetic Foot ◽  
Biofilm Formation ◽  
Guar Gum ◽  
Dependent Manner ◽  
Accessory Gene ◽  
Diabetic Foot Infections ◽  
Subinhibitory Concentrations ◽  
Dose Dependent

A new approach to diabetic foot infections (DFIs) has been investigated, using a nisin-biogel combining the antimicrobial peptide (AMP) nisin with the natural polysaccharide guar-gum. Since in in vivo conditions bacteria may be exposed to decreased antimicrobial concentrations, known as subinhibitory concentrations (sub-MICs), effects of nisin-biogel sub-MIC values corresponding to 1/2, 1/4 and 1/8 of nisin’s minimum inhibitory concentration (MIC) on virulence expression by six Staphylococcus aureus DFI isolates was evaluated by determining bacteria growth rate; expression of genes encoding for staphylococcal protein A (spA), coagulase (coa), clumping factor A (clfA), autolysin (atl), intracellular adhesin A (icaA), intracellular adhesin D (icaD), and the accessory gene regulator I (agrI); biofilm formation; Coa production; and SpA release. Nisin-biogel sub-MICs decreased bacterial growth in a strain- and dose-dependent manner, decreased agrI, atl and clfA expression, and increased spA, coa, icaA and icaD expression. Biofilm formation increased in the presence of nisin-biogel at 1/4 and 1/8 MIC, whereas 1/2 MIC had no effect. Finally, nisin-biogel at sub-MICs did not affect coagulase production, but decreased SpA production in a dose-dependent manner. Results highlight the importance of optimizing nisin-biogel doses before proceeding to in vivo trials, to reduce the risk of virulence factor’s up-regulation due to the presence of inappropriate antimicrobial concentrations.

scholarly journals Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 810-817 ◽  
Author(s):  
KJ Balazovich ◽  
JE Smolen ◽  
LA Boxer
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Dose Dependent

Abstract Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

scholarly journals Regulation of Protein Phosphorylation and Pathogen Phagocytosis by Surfactant Protein A

1999 ◽  
Vol 67 (9) ◽  
pp. 4693-4699 ◽  
Author(s):  
Trista L. Schagat ◽  
Michael James Tino ◽  
Jo Rae Wright
Keyword(s):  
Protein Phosphorylation ◽  
Alveolar Macrophages ◽  
Actin Polymerization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Dependent Manner ◽  
Cell Functions

ABSTRACT Surfactant protein A (SP-A), a pulmonary member of the collectin family of proteins, facilitates the rapid clearance of pathogens by upregulating immune cell functions in the lungs. SP-A binds to bacteria and targets them for rapid phagocytosis by alveolar macrophages, but the mechanism by which this stimulation occurs is not clear. To characterize the intracellular events that may be involved, we examined the roles of protein phosphorylation and cytoskeletal polymerization in SP-A-stimulated phagocytosis. In rat alveolar macrophages, SP-A stimulated rapid tyrosine phosphorylation of specific proteins in a dose- and time-dependent manner. The pattern of proteins that were phosphorylated in response to SP-A, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar to that observed for immunoglobulin G (IgG)-stimulated macrophages. Both SP-A and IgG stimulated increases in phagocytosis of Streptococcus pneumoniae above levels in the absence of added protein by 394% ± 81% and 200% ± 25%, respectively. Phagocytosis in both cases was dependent on tyrosine kinases, protein kinase C, and actin polymerization but not on microtubule activity. These studies show that SP-A utilizes pathways similar to those used by IgG to increase macrophage phagocytosis of bacteria.

scholarly journals 1202. Subinhibitory Concentrations of Omadacycline Inhibit Staphylococcus aureus Hemolytic Activity in Vitro

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S622-S623
Author(s):  
Alisa W Serio ◽  
S Ken Tanaka ◽  
Kelly Wright ◽  
Lynne Garrity-Ryan
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Synthesis ◽  
Virulence Factors ◽  
Hemolytic Activity ◽  
Protein Biosynthesis ◽  
The United States ◽  
Aureus Strain ◽  
Protein Synthesis Inhibitors ◽  
Subinhibitory Concentrations

Abstract Background In animal models of Staphylococcus aureus infection, α-hemolysin has been shown to be a key virulence factor. Treatment of S. aureus with subinhibitory levels of protein synthesis inhibitors can decrease α-hemolysin expression. Omadacycline, a novel aminomethylcycline antibiotic in the tetracycline class of bacterial protein biosynthesis inhibitors, is approved in the United States for treatment of community-acquired bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI) in adults. This study was performed to determine the durability of inhibition and effect of subinhibitory concentrations of omadacycline on S. aureus hemolytic activity. Methods All experiments used the methicillin-sensitive S. aureus strain Wood 46 (ATCC 10832), a laboratory strain known to secrete high levels of α-hemolysin. Minimum inhibitory concentrations (MICs) of omadacycline and comparator antibiotics (tetracycline, cephalothin, clindamycin, vancomycin, linezolid) were determined. Growth of S. aureus with all antibiotics was determined and the percentage of hemolysis assayed. “Washout” experiments were performed with omadacycline only. Results S. aureus cultures treated with 1/2 or 1/4 the MIC of omadacycline for 4 hours showed hemolysis units/108 CFU of 47% and 59% of vehicle-treated cultures, respectively (Fig. 1A, 1B). In washout experiments, treatment with as little as 1/4 the MIC of omadacycline for 1 hour decreased the hemolysis units/108 CFU by 60% for 4 hours following removal of the drug (Table 1). Figure 1 Table 1 Conclusion Omadacycline inhibited S. aureus hemolytic activity in vitro at subinhibitory concentrations and inhibition was maintained for ≥ 4 hours after removal of extracellular drug (Fig. 2). The suppression of virulence factors throughout the approved omadacycline dosing interval, in addition to the in vitro potency of omadacycline, may contribute to the efficacy of omadacycline for ABSSSI and CABP due to virulent S. aureus. This finding may apply to other organisms and other virulence factors that require new protein synthesis to establish disease. Figure 2 Disclosures Alisa W. Serio, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) S. Ken Tanaka, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) Kelly Wright, PharmD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) Lynne Garrity-Ryan, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder)

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.

Affinity chromatography purification and partial characterization of phytohemagglutinin-receptor glycoproteins from porcine splenic lymphocytes

1985 ◽  
Vol 63 (9) ◽  
pp. 932-940 ◽  
Author(s):  
Gilles Dupuis ◽  
Jean-Pierre Doucet ◽  
Bânû Bastin ◽  
Jeannine Cardin
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Interleukin 2 ◽  
Lymphocyte Activation ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Murine Cell Line ◽  
Lymphocyte Cultures

We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA – Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9–2.4% of the amount of protein applied to the column. They contained 20 ± 1.3% hexose and 1.7 ± 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50–55, 75, 95, 130, and 155 kdaltons along with minor bands of 20–40, 42, 45, 60–65, 175, and 200–250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 106 cells/mL in the presence of PHA. A 50% inhibition was observed when 20 μg/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 μg/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line. It is suggested that the affinity-purified PHA-reactive glycoproteins are inhibitors of PHA-dependent cellular responses because they compete with PHA-receptor sites on the lymphocyte plasma membrane. A mouse antiserum raised against the purified glycoproteins inhibited PHA-induced lymphocyte activation, but did not stimulate lymphocytes when added alone to lymphocyte cultures or in combination with a antimouse antiserum.

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