Luciferase Reporter Gene System to Detect Cell Wall Stress Stimulon Induction in Staphylococcus aureus

2016 ◽  
pp. 139-150 ◽  
Author(s):  
Vanina Dengler ◽  
Nadine McCallum
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Reporter Gene ◽  
Wall Stress ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Cell Wall Stress ◽  
Reporter Gene System

scholarly journals Analysis of Promoter Function in Aspergillus fumigatus

2012 ◽  
Vol 11 (9) ◽  
pp. 1167-1177 ◽  
Author(s):  
Sanjoy Paul ◽  
J. Stacey Klutts ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Reporter Gene ◽  
Reporter Genes ◽  
Antifungal Drugs ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Gene System ◽  
Reporter Gene System

ABSTRACTThe filamentous fungusAspergillus fumigatusis an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to controlA. fumigatushas led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. InA. fumigatus, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs inA. fumigatusis referred to ascyp51A. In order to dissect transcription ofcyp51Atranscription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use inA. fumigatus. These reporter genes can either replicate autonomously or be targeted to thepyrGlocus, generating an easily assayable uracil auxotrophy. We fused eight differentA. fumigatuspromoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in thecyp51Agene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression inA. fumigatus.

scholarly journals Mutational Analyses of Open Reading Frames within thevraSROperon and Their Roles in the Cell Wall Stress Response ofStaphylococcus aureus

2011 ◽  
Vol 55 (4) ◽  
pp. 1391-1402 ◽  
Author(s):  
N. McCallum ◽  
P. Stutzmann Meier ◽  
R. Heusser ◽  
B. Berger-Bächi
Keyword(s):  
Signal Transduction ◽  
Cell Wall ◽  
Cell Envelope ◽  
Wall Stress ◽  
Integral Membrane Protein ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Cell Wall Stress ◽  
Type Strains

ABSTRACTThe exposure ofStaphylococcus aureusto a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. ThevraSRgenes form part of the four-cistron autoregulatory operonorf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed thatorf1played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF inS. aureusappears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.

Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation

1997 ◽  
Vol 2 (4) ◽  
pp. 235-240 ◽  
Author(s):  
Samantha E. George ◽  
Peter J. Bungay ◽  
Louise H. Naylor
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Functional Assay ◽  
Ionophore A23187 ◽  
Camp Accumulation ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Gene Assay ◽  
Reporter Gene System

A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

Differential Effects of Components in Artemisia annua Extract on the Induction of Drug-Metabolizing Enzyme Expression Mediated by Nuclear Receptors

Planta Medica ◽  
2020 ◽  
Vol 86 (12) ◽  
pp. 867-875
Author(s):  
Xueli Zhang ◽  
Ran Meng ◽  
Haina Wang ◽  
Jie Xing
Keyword(s):  
Reporter Gene ◽  
Artemisia Annua ◽  
Constitutive Androstane Receptor ◽  
Pregnane X Receptor ◽  
Luciferase Reporter ◽  
Induction Effect ◽  
Luciferase Reporter Gene ◽  
Gene System ◽  
Drug Metabolizing Enzyme ◽  
Reporter Gene System

Abstract Artemisia annua tea is a popular dosage form used to treat and prevent malaria in some developing countries. However, repeated drinking leads to an obviously decreased efficacy, which may be related to the induction of metabolizing enzymes by artemisinin. In the present study, the ability of different components in A. annua to activate the pregnane X receptor and constitutive androstane receptor was evaluated by the dual luciferase reporter gene system. The changes in mRNA and protein expression of CYP3A4 and CYP2B6 were determined by quantitative real-time PCR and Western blotting. Results showed that in the pregnane X receptor-mediated CYP3A4 reporter gene system, chrysosplenetin and arteannuin B exhibited a weak induction effect on pregnane X receptor wt, while arteannuin A had a strong induction effect on pregnane X receptor wt and pregnane X receptor 370 and a weak induction effect on pregnane X receptor 163. In the pregnane X receptor-mediated CYP2B6 reporter gene system, arteannuin A had a moderate induction effect on pregnane X receptor wt and pregnane X receptor 379, and a weak induction effect on pregnane X receptor 403, while arteannuin B had a weak induction effect on pregnane X receptor wt and pregnane X receptor 379. Arteannuin A had a strong induction effect on constitutive androstane receptor 3 in constitutive androstane receptor-mediated CYP3A4/2B6 reporter gene systems, while arteannuin B showed a weak induction effect on constitutive androstane receptor 3 in the constitutive androstane receptor-mediated CYP2B6 reporter gene system. The mRNA and protein expressions of CYP3A4 and CYP2B6 were increased when the pregnane X receptor or constitutive androstane receptor was activated. Various components present in A. annua differentially affect the activities of pregnane X receptor isoforms and the constitutive androstane receptor, which indicates the possibility of a drug-drug interaction. This partly explains the decline in efficacy after repeated drinking of A. annua tea.

scholarly journals Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents

1995 ◽  
Vol 309 (2) ◽  
pp. 385-387 ◽  
Author(s):  
O Benzakour ◽  
C Kanthou ◽  
U Dennehy ◽  
A al Haq ◽  
L P Berg ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Luciferase Activity ◽  
Primary Cultures ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Elements ◽  
Gene System ◽  
Reporter Gene System

The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2′-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.

scholarly journals Small Alarmone Synthetases RelP and RelQ of Staphylococcus aureus Are Involved in Biofilm Formation and Maintenance Under Cell Wall Stress Conditions

2020 ◽  
Vol 11 ◽  
Author(s):  
Andrea Salzer ◽  
Daniela Keinhörster ◽  
Christina Kästle ◽  
Benjamin Kästle ◽  
Christiane Wolz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Biofilm Formation ◽  
Wall Stress ◽  
Stress Conditions ◽  
Cell Wall Stress
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