Research

15 pages, 2431 KiB

Open AccessArticle

Exploring Cryptococcus neoformans CYP51 and Its Cognate Reductase as a Drug Target

by Yasmeen N. Ruma, Mikhail V. Keniya and Brian C. Monk

J. Fungi 2022, 8(12), 1256; https://doi.org/10.3390/jof8121256 - 28 Nov 2022

Cited by 1 | Viewed by 1504

Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or [...] Read more.

Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or without its cognate hexahistidine-tagged NADPH-cytochrome P450 reductase (CnCPR-6×His), in a Saccharomyces cerevisiae host system has been used to characterise these enzymes. The heterologous expression of CnCYP51-6×His complemented deletion of the host CYP51 and conferred increased susceptibility to both short-tailed and long-tailed azole drugs. In addition, co-expression of CnCPR-6×His decreased susceptibility 2- to 4-fold for short-tailed but not long-tailed azoles. Type 2 binding of azoles to CnCYP51-6×His and assay of NADPH cytochrome P450 reductase activity confirmed that the heterologously expressed CnCYP51 and CnCPR are functional. The constructs have potential as screening tools and use in structure-directed antifungal discovery. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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13 pages, 1870 KiB

Open AccessArticle

Role of Cytosolic Malic Enzyme in Oleaginicity of High-Lipid-Producing Fungal Strain Mucor circinelloides WJ11

by Abu Bakr Ahmad Fazili, Aabid Manzoor Shah, Tahira Naz, Shaista Nosheen, Wu Yang, Victoriano Garre, Younis Majeed, Mohammed Khalid Al-Sadoon and Yuanda Song

J. Fungi 2022, 8(3), 265; https://doi.org/10.3390/jof8030265 - 05 Mar 2022

Cited by 2 | Viewed by 2173

Abstract

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, [...] Read more.

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, has been found to regulate lipid accumulation in oleaginous fungi. In the present study, the role of two cytosolic ME genes, cmalA and cmalB, in the lipid accumulation of the M. circinelloides high-lipid-producing strain WJ11, was evaluated. Strains overexpressing cmalA and cmalB showed a 9.8- and 6.4-fold rise in specific ME activity, respectively, and an elevation of the lipid content by 23.2% and 5.8%, respectively, suggesting that these genes are involved in lipid biosynthesis. Due to increased lipid accumulation, overall GLA content in biomass was observed to be elevated by 11.42% and 16.85% in cmalA and cmalB overexpressing strains, respectively. Our study gives an important insight into different studies exploring the role of the cmalA gene, while we have for the first time investigated the role of the cmalB gene in the M. circinelloides WJ11 strain. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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17 pages, 2750 KiB

Open AccessArticle

Filament Negative Regulator CDC4 Suppresses Glycogen Phosphorylase Encoded GPH1 That Impacts the Cell Wall-Associated Features in Candida albicans

by Wei-Chung Lai, Hsiao-Chi Hsu, Chun-Wen Cheng, Shao-Hung Wang, Wan Chen Li, Po-Szu Hsieh, Tzu-Ling Tseng, Ting-Hui Lin and Jia-Ching Shieh

J. Fungi 2022, 8(3), 233; https://doi.org/10.3390/jof8030233 - 26 Feb 2022

Viewed by 2240

Abstract

We have previously identified Candida albicans GPH1 (orf19.7021) whose protein product was associated with C. albicans Cdc4. The GPH1 gene is a putative glycogen phosphorylase because its Saccharomyces cerevisiae homolog participates in glycogen catabolism, which involves the synthesis of β-glucan of the fungal [...] Read more.

We have previously identified Candida albicans GPH1 (orf19.7021) whose protein product was associated with C. albicans Cdc4. The GPH1 gene is a putative glycogen phosphorylase because its Saccharomyces cerevisiae homolog participates in glycogen catabolism, which involves the synthesis of β-glucan of the fungal cell wall. We made a strain whose CaCDC4 expression is repressed, and GPH1 is constitutively expressed. We established a GPH1 null mutant strain and used it to conduct the in vitro virulence assays that detect cell wall function. The in vitro virulence assay is centered on biofilm formation in which analytic procedures are implemented to evaluate cell surface hydrophobicity; competence, either in stress resistance, germ tube formation, or fibronection association; and the XTT-based adhesion and biofilm formation. We showed that the constitutively expressed GPH1 partially suppresses filamentation when the CaCDC4 expression is repressed. The C. albicans Gph1 protein is reduced in the presence of CaCdc4 in comparison with the absence of CaCdc4. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant displayed a reduction in the capability to form germ tubes and the cell surface hydrophobicity but an increase in binding with fibronectin. Compared with the wild-type strain, the gph1Δ/gph1Δ mutant showed a rise in adhesion, the initial stage of biofilm formation, but displayed a similar capacity to form a mature biofilm. There was no major impact on the gph1Δ/gph1Δ mutant regarding the conditions of cell wall damaging and TOR pathway-associated nutrient depletion. We conclude that GPH1, adversely regulated by the filament suppressor CDC4, contributes to cell wall function in C. albicans. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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20 pages, 4411 KiB

Open AccessEditor’s ChoiceArticle

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

by Yasmeen N. Ruma, Mikhail V. Keniya, Joel D. A. Tyndall and Brian C. Monk

J. Fungi 2022, 8(1), 69; https://doi.org/10.3390/jof8010069 - 10 Jan 2022

Cited by 11 | Viewed by 2507

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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15 pages, 5167 KiB

Open AccessArticle

Transcription Factor MaMsn2 Regulates Conidiation Pattern Shift under the Control of MaH1 through Homeobox Domain in Metarhizium acridum

by Dongxu Song, Yueqing Cao and Yuxian Xia

J. Fungi 2021, 7(10), 840; https://doi.org/10.3390/jof7100840 - 07 Oct 2021

Cited by 6 | Viewed by 1747

Abstract

The growth pattern of filamentous fungi can switch between hyphal radial polar growth and non-polar yeast-like cell growth depending on the environmental conditions. Asexual conidiation after radial polar growth is called normal conidiation (NC), while yeast-like cell growth is called microcycle conidiation (MC). [...] Read more.

The growth pattern of filamentous fungi can switch between hyphal radial polar growth and non-polar yeast-like cell growth depending on the environmental conditions. Asexual conidiation after radial polar growth is called normal conidiation (NC), while yeast-like cell growth is called microcycle conidiation (MC). Previous research found that the disruption of MaH1 in Metarhizium acridum led to a conidiation shift from NC to MC. However, the regulation mechanism is not clear. Here, we found MaMsn2, an Msn2 homologous gene in M. acridum, was greatly downregulated when MaH1 was disrupted (ΔMaH1). Loss of MaMsn2 also caused a conidiation shift from NC to MC on a nutrient-rich medium. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that MaH1 could bind to the promoter region of the MaMsn2 gene. Disrupting the interaction between MaH1 and the promoter region of MaMsn2 significantly downregulated the transcription level of MaMsn2, and the overexpression of MaMsn2 in ΔMaH1 could restore NC from MC of ΔMaH1. Our findings demonstrated that MaMsn2 played a role in maintaining the NC pattern directly under the control of MaH1, which revealed the molecular mechanisms that regulated the conidiation pattern shift in filamentous fungi for the first time. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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16 pages, 2413 KiB

Open AccessArticle

DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

by Macario Osorio-Concepción, Carlos Lax, Eusebio Navarro, Francisco E. Nicolás and Victoriano Garre

J. Fungi 2021, 7(9), 738; https://doi.org/10.3390/jof7090738 - 08 Sep 2021

Cited by 4 | Viewed by 2216

Abstract

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in [...] Read more.

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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Review

Jump to: Research, Other

22 pages, 1537 KiB

Open AccessReview

Recent Molecular Tools for the Genetic Manipulation of Highly Industrially Important Mucoromycota Fungi

by Hassan Mohamed, Tahira Naz, Junhuan Yang, Aabid Manzoor Shah, Yusuf Nazir and Yuanda Song

J. Fungi 2021, 7(12), 1061; https://doi.org/10.3390/jof7121061 - 10 Dec 2021

Cited by 6 | Viewed by 3159

Abstract

Mucorales is the largest and most well-studied order of the phylum Mucormycota and is known for its rapid growth rate and various industrial applications. The Mucorales fungi are a fascinating group of filamentous organisms with many uses in research and the industrial and [...] Read more.

Mucorales is the largest and most well-studied order of the phylum Mucormycota and is known for its rapid growth rate and various industrial applications. The Mucorales fungi are a fascinating group of filamentous organisms with many uses in research and the industrial and medical fields. They are widely used biotechnological producers of various secondary metabolites and other value-added products. Certain members of Mucorales are extensively used as model organisms for genetic and molecular investigation and have extended our understanding of the metabolisms of other members of this order as well. Compared with other fungal species, our understanding of Mucoralean fungi is still in its infancy, which could be linked to their lack of effective genetic tools. However, recent advancements in molecular tools and approaches, such as the construction of recyclable markers, silencing vectors, and the CRISPR-Cas9-based gene-editing system, have helped us to modify the genomes of these model organisms. Multiple genetic modifications have been shown to generate valuable products on a large scale and helped us to understand the morphogenesis, basic biology, pathogenesis, and host–pathogen interactions of Mucoralean fungi. In this review, we discuss various conventional and modern genetic tools and approaches used for efficient gene modification in industrially important members of Mucorales. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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Other

Jump to: Research, Review

15 pages, 2852 KiB

Open AccessProtocol

Agrobacterium tumefaciens-Mediated Transformation of NHEJ Mutant Aspergillus nidulans Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers

by Virginia Casado-del Castillo, Andrew P. MacCabe and Margarita Orejas

J. Fungi 2021, 7(11), 961; https://doi.org/10.3390/jof7110961 - 12 Nov 2021

Cited by 3 | Viewed by 2377

Abstract

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this [...] Read more.

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans. Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the ΔnkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants. Full article

(This article belongs to the Special Issue Genetic Manipulation of Fungal Model Organisms)

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