Genetically encoded fluorescence screening probe for MgrA, a global regulator in Staphylococcus aureus

RSC Advances ◽  
2015 ◽  
Vol 5 (106) ◽  
pp. 87216-87220 ◽  
Author(s):  
Yujie Wang ◽  
Hong Zhang ◽  
Qingzhou Zhang ◽  
Yujie Liang ◽  
Lin Ma ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Chinese Herb ◽  
Screening System ◽  
Global Regulator ◽  
Fluorescent Response ◽  
Screening Platform ◽  
Herb Extracts

Herein, a novel cell-based fluorescent response screening system for MgrA inhibitor selection was constructed. And this screening platform was applied for Chinese herb extracts screening with two extracts identified from 351 Chinese herb extracts.

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 Optimization of a High-Throughput 384-Well Plate-Based Screening Platform with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15442 Biofilms

2020 ◽  
Vol 21 (9) ◽  
pp. 3034 ◽  
Author(s):  
Shella Gilbert-Girard ◽  
Kirsi Savijoki ◽  
Jari Yli-Kauhaluoma ◽  
Adyary Fallarero
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Rapid Screening ◽  
Bacterial Biofilms ◽  
Main Concern ◽  
Assay Optimization ◽  
Screening Platform

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

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 Spx Is a Global Effector Impacting Stress Tolerance and Biofilm Formation in Staphylococcus aureus

2006 ◽  
Vol 188 (13) ◽  
pp. 4861-4870 ◽  
Author(s):  
Sünje Johanna Pamp ◽  
Dorte Frees ◽  
Susanne Engelmann ◽  
Michael Hecker ◽  
Hanne Ingmer
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Thioredoxin Reductase ◽  
Growth Conditions ◽  
Redox Status ◽  
Global Regulator ◽  
Two Dimensional Gel Electrophoresis ◽  
Growth Defects ◽  
Stress Protection ◽  
Wide Range

ABSTRACT In Bacillus subtilis, Spx was recently characterized as a novel type of global regulator whose activity is regulated by the redox status of the cells. In the present study, we demonstrate that inactivation of Spx in the important pathogen Staphylococcus aureus renders the cells hypersensitive to a wide range of stress conditions including high and low temperature, high osmolarity, and hydrogen peroxide. Moreover, growth was restricted under nonstress conditions. Two-dimensional gel electrophoresis revealed that the proteome of the spx mutant differs substantially from the proteome of wild-type cells, supporting the finding that Spx is also a global regulator in S. aureus. More specifically, we demonstrated that Spx is required for transcription of trxB, encoding thioredoxin reductase, under all growth conditions examined. As trxB is essential in S. aureus, we speculate that the severely reduced trxB transcription could account for some of the growth defects of the spx mutant. Inactivation of spx also enhanced biofilm formation. S. aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin encoded by the ica operon. Interestingly, our data indicate that the augmented capacity of the spx mutant to form biofilms is due to Spx modulating the expression of icaR, encoding a repressor of the structural ica genes (icaABCD). In summary, we conclude that Spx fulfills an important role for growth, general stress protection, and biofilm formation in S. aureus.

scholarly journals RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA

2015 ◽  
Vol 112 (45) ◽  
pp. 14036-14041 ◽  
Author(s):  
Ravi Kr. Gupta ◽  
Thanh T. Luong ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Biological Effects ◽  
Virulence Gene ◽  
Regulatory Function ◽  
Stable Complex ◽  
Global Regulator ◽  
Sensing System ◽  
Quorum Sensing System ◽  
P2 Promoter ◽  
Virulence Gene Regulation

RNAIII, the effector of the agr quorum-sensing system, plays a key role in virulence gene regulation in Staphylococcus aureus, but how RNAIII transcriptionally regulates its downstream genes is not completely understood. Here, we show that RNAIII stabilizes mgrA mRNA, thereby increasing the production of MgrA, a global transcriptional regulator that affects the expression of many genes. The mgrA gene is transcribed from two promoters, P1 and P2, to produce two mRNA transcripts with long 5′ UTR. Two adjacent regions of the mgrA mRNA UTR transcribed from the upstream P2 promoter, but not the P1 promoter, form a stable complex with two regions of RNAIII near the 5′ and 3′ ends. We further demonstrate that the interaction has several biological effects. We propose that MgrA can serve as an intermediary regulator through which agr exerts its regulatory function.

In vitro Effect of Chinese Herb Extracts on Caries-Related Bacteria and Glucan

2008 ◽  
Vol 25 (4) ◽  
pp. 236-239 ◽  
Author(s):  
Ming Yu Li ◽  
Zheng Liu
Keyword(s):  
Oral Care ◽  
Chinese Herb ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Sensitivity Tests ◽  
Glucan Synthesis ◽  
Bacterial Sensitivity ◽  
Vitro Effect ◽  
Herb Extracts

The purpose of this study was to evaluate the inhibitory effects of herb extracts on caries -related bacteria and glucan of dental plaque in vitro. Bacterial sensitivity tests were done using broth dilution, and the phenol sulphate method was used to assess glucan inhibition. The results showed that tannic acid could inhibit bacterial growth more effectively than other herb extracts. Eugenol showed a 46.87 ± 12.74 and 36.67 ± 6.30 % inhibitory effect on insoluble and soluble glucan synthesis, respectively. Cnidium, barbaloin, caryophyllin, and piperine had > 40.0 % inhibitory effect on soluble glucan synthesis. Both insoluble and soluble glucan synthesis of S. sobrinus were inhibited by eugenol and piperine. Eugenol and piperine were efficacious in inhibiting glucan synthesis making them desirable agents for oral care products.

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