scholarly journals Characterisation of Candida parapsilosis CYP51 as a Drug Target Using Saccharomyces cerevisiae as Host

2022 ◽  
Vol 8 (1) ◽  
pp. 69
Author(s):  
Yasmeen N. Ruma ◽  
Mikhail V. Keniya ◽  
Joel D. A. Tyndall ◽  
Brian C. Monk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome P450 ◽  
Candida Parapsilosis ◽  
Constitutive Expression ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Functional Studies ◽  
Opportunistic Fungal Pathogen ◽  
Clinically Significant ◽  
Binding Spectra

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery.

scholarly journals Hospital outbreak of fluconazole-resistant Candida parapsilosis: arguments for clonal transmission and long-term persistence

2021 ◽  
Author(s):  
Arnaud Fekkar ◽  
Marion Blaize ◽  
Adrien Bouglé ◽  
Anne-Cécile Normand ◽  
Audrey Raoelina ◽  
...  
Keyword(s):  
Candida Parapsilosis ◽  
Pathogenic Fungi ◽  
Epidemiological Survey ◽  
Multidrug Resistant ◽  
Antifungal Drugs ◽  
Hospital Outbreak ◽  
Included Review ◽  
Invasive Infections ◽  
Clonal Spread ◽  
Overall Mortality

The worldwide emergence of multidrug-resistant pathogenic fungi is a threat to human health. At this very moment, an emergence of Candida parapsilosis isolates harbouring a resistance to fluconazole, one of the most popular antifungal drugs, is being described in several countries. We seek to better understanding the epidemiology, pathogenicity and transmission of resistant Candida parapsilosis. Faced with an outbreak of invasive infections due to resistant isolates of C. parapsilosis, we performed a 7-year retrospective and prospective analysis of 283 C. parapsilosis isolates collected in 240 patients, among who 111 had invasive candidiasis. Study included review of hospital records, genotyping analysis and susceptibility testing that allow determining the type and outcome of infections, as well as the spatial and temporal spread of clusters. Overall the incidence of azole resistance was 7.5%. Genotyping analysis unveiled several previously undetected outbreaks and clonal spread of susceptible and resistant isolates over a long period of time. In comparison with susceptible isolates, resistant ones have a more restricted genetic diversity and seem to be more likely to spread and more frequently associated with invasive infections. In intensive care units, patients with invasive infections due to resistant isolates had poorer outcome (overall mortality at day 30 of 40%; 4/10) than susceptible ones (overall mortality at day 30 of 26.5%; 9/34). Our results suggest that the propensity of C. parapsilosis to spread on an epidemic fashion is underestimated, which warrants reinforced control and epidemiological survey of this species.

The Regulatory Function of the Molecular Chaperone Hsp90 in the Cell Wall Integrity of Pathogenic Fungi

2018 ◽  
Vol 16 (1) ◽  
pp. 44-53
Author(s):  
Marina Campos Rocha ◽  
Camilla Alves Santos ◽  
Iran Malavazi
Keyword(s):  
Cell Wall ◽  
Antifungal Therapy ◽  
Molecular Chaperone ◽  
Regulatory Protein ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Cell Wall Integrity ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Hsp90 Inhibition

Different signaling cascades including the Cell Wall Integrity (CWI), the High Osmolarity Glycerol (HOG) and the Ca2+/calcineurin pathways control the cell wall biosynthesis and remodeling in fungi. Pathogenic fungi, such as Aspergillus fumigatus and Candida albicans, greatly rely on these signaling circuits to cope with different sources of stress, including the cell wall stress evoked by antifungal drugs and the host’s response during infection. Hsp90 has been proposed as an important regulatory protein and an attractive target for antifungal therapy since it stabilizes major effector proteins that act in the CWI, HOG and Ca2+/calcineurin pathways. Data from the human pathogen C. albicans have provided solid evidence that loss-of-function of Hsp90 impairs the evolution of resistance to azoles and echinocandin drugs. In A. fumigatus, Hsp90 is also required for cell wall integrity maintenance, reinforcing a coordinated function of the CWI pathway and this essential molecular chaperone. In this review, we focus on the current information about how Hsp90 impacts the aforementioned signaling pathways and consequently the homeostasis and maintenance of the cell wall, highlighting this cellular event as a key mechanism underlying antifungal therapy based on Hsp90 inhibition.

scholarly journals Regulatory Mechanisms Controlling Expression of the DAN/TIR Mannoprotein Genes During Anaerobic Remodeling of the Cell Wall in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1169-1177
Author(s):  
Natalia E Abramova ◽  
Brian D Cohen ◽  
Odeniel Sertil ◽  
Rachna Kapoor ◽  
Kelvin J A Davies ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Consensus Sequence ◽  
Ectopic Expression ◽  
Constitutive Expression ◽  
Anaerobic Growth ◽  
Zinc Cluster ◽  
Sterol Transport ◽  
Genes Encoding ◽  
Altered Regulation ◽  
Repression Domain

Abstract The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Δ allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

scholarly journals The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

2021 ◽  
Vol 7 (2) ◽  
pp. 86
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

scholarly journals Antimicrobial Activity of Antifungal Drugs on Candida Parapsilosis Studied by Scanning Ionconductance Microscopy (SICM)

2021 ◽  
Vol 120 (3) ◽  
pp. 361a
Author(s):  
Nikita Savin ◽  
Vasilii Kolmogorov ◽  
Aleksei Iakovlev ◽  
Roman Timoshenko ◽  
Petr Gorelkin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Candida Parapsilosis ◽  
Antifungal Drugs

scholarly journals CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

2004 ◽  
Vol 48 (12) ◽  
pp. 4505-4512 ◽  
Author(s):  
Chia-Geun Chen ◽  
Yun-Liang Yang ◽  
Hsin-I Shih ◽  
Chia-Li Su ◽  
Hsiu-Jung Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Type Strain ◽  
Antifungal Agents ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Galactosidase Activity ◽  
Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

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