scholarly journals Pivotal Role for a Tail Subunit of the RNA Polymerase II Mediator Complex CgMed2 in Azole Tolerance and Adherence in Candida glabrata

2014 ◽  
Vol 58 (10) ◽  
pp. 5976-5986 ◽  
Author(s):  
Sapan Borah ◽  
Raju Shivarathri ◽  
Vivek Kumar Srivastava ◽  
Sélène Ferrari ◽  
Dominique Sanglard ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Candida Glabrata ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Pivotal Role ◽  
Mediator Complex ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Azole Antifungals

ABSTRACTAntifungal therapy failure can be associated with increased resistance to the employed antifungal agents.Candida glabrata, the second most common cause of invasive candidiasis, is intrinsically less susceptible to the azole class of antifungals and accounts for 15% of allCandidabloodstream infections. Here, we show thatC. glabrataMED2(CgMED2), which codes for a tail subunit of the RNA polymerase II Mediator complex, is required for resistance to azole antifungal drugs inC. glabrata. An inability to transcriptionally activate genes encoding a zinc finger transcriptional factor, CgPdr1, and multidrug efflux pump, CgCdr1, primarily contributes to the elevated susceptibility of theCgmed2Δ mutant toward azole antifungals. We also report for the first time that theCgmed2Δ mutant exhibits sensitivity to caspofungin, a constitutively activated protein kinase C-mediated cell wall integrity pathway, and elevated adherence to epithelial cells. The increased adherence of theCgmed2Δ mutant was attributed to the elevated expression of theEPA1andEPA7genes. Further, our data demonstrate thatCgMED2is required for intracellular proliferation in human macrophages and modulates survival in a murine model of disseminated candidiasis. Lastly, we show an essential requirement for CgMed2, along with the Mediator middle subunit CgNut1 and the Mediator cyclin-dependent kinase/cyclin subunit CgSrb8, for the high-level fluconazole resistance conferred by the hyperactive allele of CgPdr1. Together, our findings underscore a pivotal role for CgMed2 in basal tolerance and acquired resistance to azole antifungals.

scholarly journals Transcription Factor ADS-4 Regulates Adaptive Responses and Resistance to Antifungal Azole Stress

2015 ◽  
Vol 59 (9) ◽  
pp. 5396-5404 ◽  
Author(s):  
Kangji Wang ◽  
Zhenying Zhang ◽  
Xi Chen ◽  
Xianyun Sun ◽  
Cheng Jin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Bzip Transcription Factor ◽  
Azole Resistance ◽  
Homologous Gene ◽  
Adaptive Responses ◽  
Transcriptional Responses ◽  
Content Type

ABSTRACTAzoles are commonly used as antifungal drugs or pesticides to control fungal infections in medicine and agriculture. Fungi adapt to azole stress by rapidly activating the transcription of a number of genes, and transcriptional increases in some azole-responsive genes can elevate azole resistance. The regulatory mechanisms that control transcriptional responses to azole stress in filamentous fungi are not well understood. This study identified a bZIP transcription factor, ADS-4 (antifungaldrugsensitive-4), as a new regulator of adaptive responses and resistance to antifungal azoles. Transcription ofads-4inNeurospora crassacells increased when they were subjected to ketoconazole treatment, whereas the deletion ofads-4resulted in hypersensitivity to ketoconazole and fluconazole. In contrast, the overexpression ofads-4increased resistance to fluconazole and ketoconazole inN. crassa. Transcriptome sequencing (RNA-seq) analysis, followed by quantitative reverse transcription (qRT)-PCR confirmation, showed that ADS-4 positively regulated the transcriptional responses of at least six genes to ketoconazole stress inN. crassa. The gene products of four ADS-4-regulated genes are known contributors to azole resistance, including the major efflux pump CDR4 (Pdr5p ortholog), an ABC multidrug transporter (NcAbcB), sterol C-22 desaturase (ERG5), and a lipid transporter (NcRTA2) that is involved in calcineurin-mediated azole resistance. Deletion of theads-4-homologous gene Afads-4inAspergillus fumigatuscaused hypersensitivity to itraconazole and ketoconazole, which suggested that ADS-4 is a functionally conserved regulator of adaptive responses to azoles. This study provides important information on a new azole resistance factor that could be targeted by a new range of antifungal pesticides and drugs.

scholarly journals Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Meinan Lyu ◽  
Mitchell A. Moseng ◽  
Jennifer L. Reimche ◽  
Concerta L. Holley ◽  
Vijaya Dhulipala ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drug Resistance ◽  
Neisseria Gonorrhoeae ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transmitted Infection ◽  
Content Type ◽  
Cryo Electron Microscopy

ABSTRACT Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

scholarly journals The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes

2014 ◽  
Vol 26 (1) ◽  
pp. 465-484 ◽  
Author(s):  
Piers A. Hemsley ◽  
Charlotte H. Hurst ◽  
Ewon Kaliyadasa ◽  
Rebecca Lamb ◽  
Marc R. Knight ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mediator Complex

scholarly journals Novel critical role of a human Mediator complex for basal RNA polymerase II transcription

EMBO Reports ◽  
2001 ◽  
Vol 2 (9) ◽  
pp. 808-813 ◽  
Author(s):  
Gerhard Mittler ◽  
Elisabeth Kremmer ◽  
H Th. Marc Timmers ◽  
Michael Meisterernst
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Mediator Complex ◽  
Rna Polymerase Ii Transcription

scholarly journals Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Shannon R. Coleman ◽  
Travis Blimkie ◽  
Reza Falsafi ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Iron Acquisition ◽  
Polymyxin B ◽  
Rna Seq ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

scholarly journals Caenorhabditis elegans as a Model System To Assess Candida glabrata, Candida nivariensis, and Candida bracarensis Virulence and Antifungal Efficacy

2020 ◽  
Vol 64 (10) ◽  
Author(s):  
Ainara Hernando-Ortiz ◽  
Estibaliz Mateo ◽  
Marcelo Ortega-Riveros ◽  
Iker De-la-Pinta ◽  
Guillermo Quindós ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Candida Glabrata ◽  
Model System ◽  
Antifungal Drugs ◽  
Phenotypic Traits ◽  
Content Type ◽  
C Elegans ◽  
Host Fungus ◽  
Candida Bracarensis ◽  
Candida Nivariensis

ABSTRACT Although Candida albicans remains the major etiological agent of invasive candidiasis, Candida glabrata and other emerging species of Candida are increasingly isolated. This species is the second most prevalent cause of candidiasis in many regions of the world. However, clinical isolates of Candida nivariensis and Candida bracarensis can be misidentified and are underdiagnosed due to phenotypic traits shared with C. glabrata. Little is known about the two cryptic species. Therefore, pathogenesis studies are needed to understand their virulence traits and their susceptibility to antifungal drugs. The susceptibility of Caenorhabditis elegans to different Candida species makes this nematode an excellent model for assessing host-fungus interactions. We evaluated the usefulness of C. elegans as a nonconventional host model to analyze the virulence of C. glabrata, C. nivariensis, and C. bracarensis. The three species caused candidiasis, and the highest virulence of C. glabrata was confirmed. Furthermore, we determined the efficacy of current antifungal drugs against the infection caused by these species in the C. elegans model. Amphotericin B and azoles showed the highest activity against C. glabrata and C. bracarensis infections, while echinocandins were more active for treating those caused by C. nivariensis. C. elegans proved to be a useful model system for assessing the pathogenicity of these closely related species.

scholarly journals SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

2014 ◽  
Vol 58 (9) ◽  
pp. 5102-5110 ◽  
Author(s):  
Bernardo Ramírez-Zavala ◽  
Selene Mogavero ◽  
Eva Schöller ◽  
Christoph Sasse ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

scholarly journals Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

2015 ◽  
Vol 36 (5) ◽  
pp. 820-831 ◽  
Author(s):  
Jürgen Niesser ◽  
Felix R. Wagner ◽  
Dirk Kostrewa ◽  
Wolfgang Mühlbacher ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Hydrophobic Pocket ◽  
Pol Ii ◽  
Content Type ◽  
Open Conformation ◽  
Peptide Release ◽  
Hydrophobic Peptide

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, theSaccharomyces cerevisiaeenzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

scholarly journals A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Jack R. Davison ◽  
Katheryn M. Lohith ◽  
Xiaoning Wang ◽  
Kostyantyn Bobyk ◽  
Sivakoteswara R. Mandadapu ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Bacterial Strains ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Membranes ◽  
Development Of Resistance ◽  
Antibiotic Compounds ◽  
Blasticidin S

ABSTRACT The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

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