scholarly journals Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

2014 ◽  
Vol 80 (19) ◽  
pp. 6154-6166 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Claire M. Hull ◽  
Nicola J. Rolley ◽  
Josie E. Parker ◽  
W. David Nes ◽  
...  
Keyword(s):  
Antifungal Agents ◽  
Leisure Activities ◽  
Binding Affinities ◽  
Farmed Fish ◽  
Gene Encoding ◽  
Content Type ◽  
Saprolegnia Ferax ◽  
Saprolegnia Parasitica ◽  
Oomycete Pathogen

ABSTRACTA candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogenSaprolegnia parasitica(SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed inEscherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibitingS. parasitica,Saprolegnia diclina, andSaprolegnia feraxgrowth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibitSaprolegniagrowth (MIC100, >256 μg ml−1). The remaining azoles inhibitedSaprolegniagrowth only at elevated concentrations (MIC100[the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml−1) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml−1). Sterol profiles of azole-treatedSaprolegniaspecies confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth ofSaprolegniaspeciesin vitro(MIC100, ∼1 to 2 μg ml−1) suggest that clotrimazole could be used againstSaprolegniainfections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.

scholarly journals Antifungal Activity of SCY-078 and Standard Antifungal Agents against 178 Clinical Isolates of Resistant and Susceptible Candida Species

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Wiley A. Schell ◽  
A. M. Jones ◽  
Katyna Borroto-Esoda ◽  
Barbara D. Alexander
Keyword(s):  
Candida Glabrata ◽  
Antifungal Agents ◽  
Candida Parapsilosis ◽  
Candida Krusei ◽  
Wild Type ◽  
Content Type ◽  
Clinical Resistance ◽  
Excellent Activity ◽  
Candida Lusitaniae

ABSTRACT SCY-078 in vitro activity was determined for 178 isolates of resistant or susceptible Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitaniae, and Candida parapsilosis, including 44 Candida isolates with known genotypic (FKS1 or FKS2 mutations), phenotypic, or clinical resistance to echinocandins. Results were compared to those for anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole. SCY-078 was shown to have excellent activity against both wild-type isolates and echinocandin- and azole-resistant isolates of Candida species.

scholarly journals Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

2015 ◽  
Vol 60 (3) ◽  
pp. 1226-1233 ◽  
Author(s):  
Petros Ioannou ◽  
Aggeliki Andrianaki ◽  
Tonia Akoumianaki ◽  
Irene Kyrmizi ◽  
Nathaniel Albert ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Antifungal Agents ◽  
Fold Increase ◽  
Culture Conditions ◽  
The Other ◽  
Related Factors ◽  
Content Type

The modestin vitroactivity of echinocandins againstAspergillusimplies that host-related factors augment the action of these antifungal agentsin vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against variousAspergillusspecies under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P= 0. 0005). Importantly, the enhanced activity of caspofungin againstAspergillusspp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinatingAspergillushyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin withAspergillushyphae (P< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery toAspergillushyphae.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

scholarly journals Low In Vitro Antifungal Activity of Tavaborole against Yeasts and Molds from Onychomycosis

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Mahdi Abastabar ◽  
Iman Haghani ◽  
Tahereh Shokohi ◽  
Mohammad Taghi Hedayati ◽  
Seyed Reza Aghili ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Antifungal Agents ◽  
Antifungal Drug ◽  
Content Type ◽  
Fungal Isolates ◽  
Yeasts And Molds ◽  
Low Activity ◽  
In Vitro Antifungal Activity

ABSTRACT The in vitro activity of tavaborole, an FDA-approved antifungal drug, was compared to that of four antifungal agents against 170 clinical fungal isolates originating from patients with onychomycosis. Tavaborole had low activity against all isolates compared to itraconazole, terbinafine, and fluconazole, the principal choices for treatment of onychomycosis. Thus, it appears that tavaborole is not a candidate for the treatment of onychomycosis due to Candida species, Aspergillus species, and dermatophytes.

scholarly journals Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Cristina Lazzarini ◽  
Krupanandan Haranahalli ◽  
Robert Rieger ◽  
Hari Krishna Ananthula ◽  
Pankaj B. Desai ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Fungal Resistance ◽  
Content Type ◽  
Highly Active ◽  
Pharmacokinetic Properties ◽  
Pass Through

ABSTRACTThe incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active againstCryptococcus neoformansin vitroand had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

scholarly journals BrucellaPeriplasmic Protein EipB Is a Molecular Determinant of Cell Envelope Integrity and Virulence

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Julien Herrou ◽  
Jonathan W. Willett ◽  
Aretha Fiebig ◽  
Daniel M. Czyż ◽  
Jason X. Cheng ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Mammalian Host ◽  
Periplasmic Protein ◽  
Periplasmic Space ◽  
Gene Encoding ◽  
Tetratricopeptide Repeats ◽  
Content Type ◽  
Family Protein ◽  
Synthetically Lethal

ABSTRACTThe Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space ofBrucellaspecies, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded β-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion ofeipBresults in defects inBrucellacell envelope integrityin vitroand in maintenance of spleen colonization in a mouse model ofBrucella abortusinfection. Transposon disruption ofttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal witheipBdeletion.ttpAis a reported virulence determinant inBrucella, and our studies ofttpAdeletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude thateipBandttpAfunction in theBrucellaperiplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host.IMPORTANCEBrucellaspecies cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequencedBrucellaand several other genera in the classAlphaproteobacteria. The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel β protein that is a determinant of cell envelope integrityin vitroand virulence in an animal model of disease.eipBgenetically interacts withttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in selectAlphaproteobacteria.

scholarly journals In Vitro Activity of Chlorhexidine Compared with Seven Antifungal Agents against 98 Fusarium Isolates Recovered from Fungal Keratitis Patients

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Claudy Oliveira dos Santos ◽  
Eva Kolwijck ◽  
Henrich A. van der Lee ◽  
Marlou C. Tehupeiory-Kooreman ◽  
Abdullah M. S. Al-Hatmi ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Antifungal Agents ◽  
Culture Collection ◽  
Fungal Keratitis ◽  
Molecular Techniques ◽  
Temperate Climate ◽  
Eye Drops ◽  
Fungal Culture ◽  
Content Type

ABSTRACT Fungal keratitis is a common but severe eye infection in tropical and subtropical areas of the world. In regions with a temperate climate, the frequency of infection is rising in patients with contact lenses and following trauma. Early and adequate therapy is important to prevent disease progression and loss of vision. The management of Fusarium keratitis is complex, and the optimal treatment is not well defined. We investigated the in vitro activity of chlorhexidine and seven antifungal agents against a well-characterized collection of Fusarium isolates recovered from patients with Fusarium keratitis. The fungus culture collection of the Center of Expertise in Mycology Radboudumc/CWZ was searched for Fusarium isolates that were cultured from cornea scrapings, ocular biopsy specimens, eye swabs, and contact lens fluid containers from patients with suspected keratitis. The Fusarium isolates that were cultured from patients with confirmed keratitis were all identified using conventional and molecular techniques. Antifungal susceptibility testing was performed according to the EUCAST broth microdilution reference method. The antifungal agents tested included amphotericin B, voriconazole, posaconazole, miconazole, natamycin, 5-fluorocytosine, and caspofungin. In addition, the activity of chlorhexidine was determined. The fungal culture collection contained 98 Fusarium isolates of confirmed fungal keratitis cases from 83 Dutch patients and 15 Tanzanian patients. The isolates were collected between 2007 and 2017. Fusarium oxysporum (n = 24, 24.5%) was the most frequently isolated species followed by Fusarium solani sensu stricto (n = 18, 18.4%) and Fusarium petroliphilum (n = 11, 11.2%). Amphotericin B showed the most favorable in vitro inhibition of Fusarium species followed by natamycin, voriconazole, and chlorhexidine, while 5-fluorocytosine, posaconazole, miconazole, and caspofungin showed no relevant inhibiting effect. However, chlorhexidine showed fungicidal activity against 90% of F. oxysporum strains and 100% of the F. solani strains. Our study supports the clinical efficacy of chlorhexidine and therefore warrants its further clinical evaluation for primary therapy of fungal keratitis, particularly in low and middle income countries where fungal keratitis is much more frequent and, currently, antifungal eye drops are often unavailable.

scholarly journals Expression of Adhesive Pili and the Collagen-Binding Adhesin Ace Is Activated by ArgR Family Transcription Factors inEnterococcus faecalis

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Dawn A. Manias ◽  
Gary M. Dunny
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Enterococcus Faecalis ◽  
Opportunistic Infections ◽  
Structural Genes ◽  
Gene Encoding ◽  
Collagen Binding ◽  
Content Type ◽  
Surface Adhesins

ABSTRACTIt was shown previously that the disruption of theahrCgene encoding a predicted ArgR family transcription factor results in a severe defect in biofilm formationin vitro, as well as a significant attenuation of virulence ofEnterococcus faecalisstrain OG1RF in multiple experimental infection models. Using transcriptome sequencing (RNA-seq), we observedahrC-dependent changes in the expression of more than 20 genes. AhrC-repressed genes included predicted determinants of arginine catabolism and several other metabolic genes and predicted transporters, while AhrC-activated genes included determinants involved in the production of surface protein adhesins. Most notably, the structural and regulatory genes of theebplocus encoding adhesive pili were positively regulated, as well as theacegene, encoding a collagen-binding adhesin. UsinglacZtranscription reporter fusions, we determined thatahrCand a secondargRtranscription factor gene,argR2, both function to activate the expression ofebpR, which directly activates the transcription of the pilus structural genes. Our data suggest that in the wild-typeE. faecalis, the low levels of EbpR limit the expression of pili and that biofilm biomass is also limited by the amount of pili expressed by the bacteria. The expression ofaceis similarly enhanced by AhrC and ArgR2, butaceexpression is not dependent on EbpR. Our results demonstrate the existence of novel regulatory cascades controlled by a pair of ArgR family transcription factors that might function as a heteromeric protein complex.IMPORTANCECell surface adhesins play critical roles in the formation of biofilms, host colonization, and the pathogenesis of opportunistic infections byEnterococcus faecalis. Here, we present new results showing that the expression of two major enterococcal surface adhesins,ebppili, and the collagen-binding protein Ace is positively regulated at the transcription level by twoargRfamily transcription factors, AhrC and ArgR2. In the case of pili, the direct target of regulation is theebpRgene, previously shown to activate the transcription of the pilus structural genes, while the activation ofacetranscription appears to be directly impacted by the two ArgR proteins. These transcription factors may represent new targets for blocking enterococcal infections.

scholarly journals In Vitro Susceptibility of Thai Pythium insidiosum Isolates to Antibacterial Agents

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Navaporn Worasilchai ◽  
Ariya Chindamporn ◽  
Rongpong Plongla ◽  
Pattama Torvorapanit ◽  
Kasama Manothummetha ◽  
...  
Keyword(s):  
Antibacterial Agents ◽  
Antifungal Agents ◽  
Radical Surgery ◽  
Synergistic Effects ◽  
In Vitro Susceptibility ◽  
Pythium Insidiosum ◽  
Content Type ◽  
Life Threatening ◽  
Report Data

ABSTRACT Human pythiosis is a life-threatening human disease caused by Pythium insidiosum. In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 μg/ml), minocycline MICs (1 to 4 μg/ml), tigecycline MICs (1 to 4 μg/ml), azithromycin MICs (1 to 16 μg/ml), and clarithromycin MICs (0.125 to 8 μg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.

scholarly journals Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

2015 ◽  
Vol 59 (8) ◽  
pp. 4707-4713 ◽  
Author(s):  
David C. Lamb ◽  
Andrew G. S. Warrilow ◽  
Nicola J. Rolley ◽  
Josie E. Parker ◽  
W. David Nes ◽  
...  
Keyword(s):  
Antifungal Agents ◽  
Acanthamoeba Castellanii ◽  
Therapeutic Agents ◽  
Effective Inhibitor ◽  
Human Pathogens ◽  
Acanthamoeba Polyphaga ◽  
Binding Studies ◽  
Content Type ◽  
Inhibition Concentration

ABSTRACTIn this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole againstAcanthamoeba castellaniiandAcanthamoeba polyphagaand assess their potential as therapeutic agents againstAcanthamoebainfections in humans. Amebicidal activities of the triazoles were assessed byin vitrominimum inhibition concentration (MIC) determinations using trophozoites ofA. castellaniiandA. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purifiedA. castellaniiCYP51 (AcCYP51) that was heterologously expressed inEscherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kdof 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibitingA. polyphagaandA. castellaniicell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibitAcanthamoebacell division (MIC, >64 mg/liter)in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation forA. castellaniiandA. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controllingAcanthamoebainfections.

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