scholarly journals Development of a Tightly Regulatable Copper-Mediated Gene Switch System in Dermatophytes

2012 ◽  
Vol 78 (15) ◽  
pp. 5204-5211 ◽  
Author(s):  
Atsushi Iwata ◽  
Mohamed Mahdi Alshahni ◽  
Yayoi Nishiyama ◽  
Koichi Makimura ◽  
Shigeru Abe ◽  
...  
Keyword(s):  
Drug Targets ◽  
Efflux Pump ◽  
Molecular Genetic ◽  
Genetic Research ◽  
Promoter Sequence ◽  
Essential Genes ◽  
Regulation System ◽  
Copper Transporter ◽  
Content Type ◽  
Targeted Gene Deletion

ABSTRACTTargeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter geneCTR4(PCTR4) and that of the copper efflux pump geneCRP1(PCRP1) derived fromTrichophyton rubrumwere examined for their response to copper inArthroderma vanbreuseghemii. PCTR4was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of PCRP1was induced by addition of copper. Importantly, PCTR4regulated the gene expression more tightly. Furthermore, when PCTR4was applied to regulate the expression of the endogenous genesERG1andTRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the PCTR4-based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

scholarly journals Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

mSystems ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Lauren E. Brooks ◽  
Sabah Ul-Hasan ◽  
Benjamin K. Chan ◽  
Mark J. Sistrom
Keyword(s):  
Global Health ◽  
Drug Targets ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Health Concerns ◽  
Efflux Pump Inhibitors ◽  
Eskape Pathogens

ABSTRACTIncreasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa, andEnterobacterspecies) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system inS. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets.IMPORTANCEIncreasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date. Our study evaluates an often-underappreciated aspect of resistance—the impact of evolutionary selection. We evaluate selection on all annotated efflux genes in all sequenced ESKAPE pathogens, providing critical context for and insight into current and future development of efflux-targeting treatments for resistant bacterial infections.

scholarly journals Recent Advances in Molecular Genetic Tools for Babesia

2021 ◽  
Vol 8 (10) ◽  
pp. 222
Author(s):  
Hassan Hakimi ◽  
Masahito Asada ◽  
Shin-ichiro Kawazu
Keyword(s):  
Drug Targets ◽  
Molecular Genetic ◽  
Essential Genes ◽  
Babesia Bovis ◽  
Bovine Babesiosis ◽  
Genetic Tools ◽  
Genome Wide ◽  
Glms Ribozyme ◽  
Novel Drug

Development of in vitro culture and completion of genome sequencing of several Babesia parasites promoted the efforts to establish transfection systems for these parasites to dissect the gene functions. It has been more than a decade since the establishment of first transfection for Babesia bovis, the causative agent of bovine babesiosis. However, the number of genes that were targeted by genetic tools in Babesia parasites is limited. This is partially due to the low efficiencies of these methods. The recent adaptation of CRISPR/Cas9 for genome editing of Babesia bovis can accelerate the efforts for dissecting this parasite’s genome and extend the knowledge on biological aspects of erythrocytic and tick stages of Babesia. Additionally, glmS ribozyme as a conditional knockdown system is available that could be used for the characterization of essential genes. The development of high throughput genetic tools is needed to dissect the function of multigene families, targeting several genes in a specific pathway, and finally genome-wide identification of essential genes to find novel drug targets. In this review, we summarized the current tools that are available for Babesia and the genes that are being targeted by these tools. This may draw a perspective for the future development of genetic tools and pave the way for the identification of novel drugs or vaccine targets.

scholarly journals Legal and ethical regulation of genetic research

2021 ◽  
Vol 25 (1) ◽  
pp. 214-231
Author(s):  
Vladimir I. Przhilenskiy
Keyword(s):  
Molecular Genetic ◽  
Genetic Research ◽  
Ethics Committees ◽  
Patient Treatment ◽  
Regulation System ◽  
Russian Society ◽  
Russian Science ◽  
Successful Development ◽  
Research Regulation ◽  
Necessary And Sufficient

The paper deals with the problems of genetic research regulation stemming from the peculiarities of this area of knowledge where research is practically inseparable from patient treatment, and cognitive practices are connected with the development and implementation of new devices, methodologies, and technologies to a much greater extent than in other areas of human activity. The issues of genetic research regulation are analyzed in the context of the concept suggested by Helga Nowotny and Giuseppe Testa distinguishing three human technologies of standardization through which control over the development of knowledge about genes is exercised. These three technologies are law, governance and bioethics, and the efficiency of their interaction is declared a necessary and sufficient condition for the successful development of molecular genetic sciences. The paper examines all the three technologies one by one, identifies the specifics and difficulties of their implementation in present-day Russian science, and assesses the genetic research prospects assuming the implementation of the aforementioned technologies. Particular attention is paid to institutionalization of ethics committees as an element of the genetic research regulation system. Substantiation is provided for the thesis that ethical committees should act as an institution combining rulemaking, management, and expert functions rather than as a structure capable of performing advisory and recommendatory functions only. The ability of the Russian society and state to withstand the grand challenges of our time is associated with the success in solving this problem.

scholarly journals Population Genetic Structure Analysis Reveals Decreased but Moderate Diversity for the Oriental Fire-Bellied Toad Introduced to Beijing after 90 Years of Independent Evolution

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1429
Author(s):  
Yang Teng ◽  
Jing Yang ◽  
Guofen Zhu ◽  
Fuli Gao ◽  
Yingying Han ◽  
...  
Keyword(s):  
Genetic Structure ◽  
Structure Analysis ◽  
Phylogenetic Trees ◽  
Molecular Genetic ◽  
Genetic Research ◽  
Haplotype Diversity ◽  
Botanical Garden ◽  
Source Population ◽  
Independent Evolution ◽  
Loop Region

Detailed molecular genetic research on amphibian populations has a significant role in understanding the genetic adaptability to local environments. The oriental fire-bellied toads (Bombina orientalis) were artificially introduced to Beijing from Shandong Province in 1927, and since then, this separated population went through an independent evolution. To explore the differentiation of the introduced population with its original population, this study analyzed the genetic structure of the oriental fire-bellied toad, based on the mitochondrial genome control region and six microsatellite sites. The results showed that the haplotype diversity and nucleotide diversity of the mitochondrial D-loop region partial sequences of the Beijing Botanical Garden population and the Baiwangshan population were lower than those of the Shangdong Kunyushan population. Microsatellite marker analysis also showed that the observed heterozygosity and expected heterozygosity of the Beijing populations were lower than those of the Kunyushan population. The phylogenetic trees and network diagrams of haplotypes indicated that the three populations were not genetically separated. However, the structure analysis showed a genetic differentiation and categorized the sampling individuals into Beijing and Shandong genetic clusters, which indicated a tendency for isolated evolution in the Beijing population. Although the Beijing populations showed a decline in genetic diversity, it was still at a moderate level, which could maintain the survival of the population. Thus, there is no need to reintroduce new individuals from the Kunyushan source population.

scholarly journals Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

2012 ◽  
Vol 56 (8) ◽  
pp. 4450-4458 ◽  
Author(s):  
Mark Veleba ◽  
Paul G. Higgins ◽  
Gerardo Gonzalez ◽  
Harald Seifert ◽  
Thamarai Schneiders
Keyword(s):  
Multidrug Resistance ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Content Type ◽  
Resistance Phenotype ◽  
Serratia Proteamaculans ◽  
Virulence Potential ◽  
Article Type ◽  
Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

scholarly journals Somatic embryo induction and Agrobacterium-mediated transformation of embryonic callus tissue in Phoebe bournei, an endangered woody species in Lauraceae

2020 ◽  
Vol 48 (2) ◽  
pp. 572-587
Author(s):  
Wenting XU ◽  
Miao ZHANG ◽  
Chen WANG ◽  
Xiongzhen LOU ◽  
Xiao HAN ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Germination Rate ◽  
Molecular Genetic ◽  
Genetic Research ◽  
Woody Species ◽  
Technical System ◽  
Transformation Rate ◽  
Original Material ◽  
Embryonic Callus ◽  
Phoebe Bournei

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation.

scholarly journals KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study

2021 ◽  
Vol 22 (1) ◽  
pp. 448
Author(s):  
Federica D’Aria ◽  
Bruno Pagano ◽  
Luigi Petraccone ◽  
Concetta Giancola
Keyword(s):  
Thermodynamic Stability ◽  
Drug Targets ◽  
Promoter Sequence ◽  
Physicochemical Study ◽  
Biological Processes ◽  
Scanning Calorimetry ◽  
Binding Properties ◽  
Anticancer Compounds ◽  
Nmr Structures ◽  
Genomic Regions

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5′-AGGGCGGTGTGGGAATAGGGAA-3′ (KRAS 22RT) and 5′-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3′ (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs.

scholarly journals Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Julia V. Bugrysheva ◽  
David Sue ◽  
Jay E. Gee ◽  
Mindy G. Elrod ◽  
Alex R. Hoffmaster ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Clavulanic Acid ◽  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Point Mutations ◽  
Comparative Genomic ◽  
Content Type ◽  
Reductase Gene ◽  
Amoxicillin Clavulanic Acid ◽  
Imipenem Resistance

ABSTRACT Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and β-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A β-lactamase and was previously implicated in resistance to β-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei. Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

scholarly journals Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible Cells

2006 ◽  
Vol 50 (11) ◽  
pp. 3597-3606 ◽  
Author(s):  
Tulika Prasad ◽  
Aparna Chandra ◽  
Chinmay K. Mukhopadhyay ◽  
Rajendra Prasad
Keyword(s):  
Multidrug Resistance ◽  
Membrane Fluidity ◽  
Iron Uptake ◽  
Efflux Pump ◽  
Disulfonic Acid ◽  
Candida Spp ◽  
Copper Transporter ◽  
Iron Depletion ◽  
Cellular Iron ◽  
Increased Sensitivity

ABSTRACT Inthis study, we show that iron depletion in Candida albicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to several drugs, including the most common antifungal, fluconazole (FLC). Several other species of Candida also displayed increased sensitivity to FLC because of iron restriction. Iron uptake mutations, namely,Δ ftr1 and Δftr2, as well as the copper transporter mutation Δccc2, which affects high-affinity iron uptake in Candida, produced increased sensitivity to FLC compared to that of the wild type. The effect of iron depletion on drug sensitivity appeared to be independent of the efflux pump proteins Cdr1p and Cdr2p. We found that iron deprivation led to lowering of membrane ergosterol by 15 to 30%. Subsequently, fluorescence polarization measurements also revealed that iron-restricted Candida cells displayed a 29 to 40% increase in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Northern blot assays revealed that the ERG11 gene was considerably down regulated in iron-deprived cells, which might account for the lowered ergosterol content. Our results show a close relationship between cellular iron and drug susceptibilities of C. albicans. Considering that multidrug resistance is a manifestation of multifactorial phenomena, the influence of cellular iron on the drug susceptibilities of Candida suggests iron as yet another novel determinant of multidrug resistance.

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