A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates

With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.

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Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery

Molecular Microbiology ◽

10.1046/j.1365-2958.2003.03697.x ◽

2003 ◽

Vol 50 (1) ◽

pp. 167-181 ◽

Cited By ~ 311

Author(s):

Terry Roemer ◽

Bo Jiang ◽

John Davison ◽

Troy Ketela ◽

Karynn Veillette ◽

...

Keyword(s):

Candida Albicans ◽

Drug Discovery ◽

Large Scale ◽

Essential Gene ◽

Antifungal Drug ◽

Gene Identification

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Synergistic Antifungal Activity of Chitosan with Fluconazole against Candida albicans, Candida tropicalis, and Fluconazole-Resistant Strains

Molecules ◽

10.3390/molecules25215114 ◽

2020 ◽

Vol 25 (21) ◽

pp. 5114

Author(s):

Wei-Hsuan Lo ◽

Fu-Sheng Deng ◽

Chih-Jung Chang ◽

Ching-Hsuan Lin

Keyword(s):

Candida Albicans ◽

Antifungal Activity ◽

Candida Tropicalis ◽

Inhibitory Effect ◽

Antifungal Drugs ◽

Drug Resistant ◽

Combinational Treatment ◽

Fluconazole Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

(1) Background: Few antifungal drugs are currently available, and drug-resistant strains have rapidly emerged. Thus, the aim of this study is to evaluate the effectiveness of the antifungal activity from a combinational treatment of chitosan with a clinical antifungal drug on Candida albicans and Candida tropicalis. (2) Methods: Minimum inhibitory concentration (MIC) tests, checkerboard assays, and disc assays were employed to determine the inhibitory effect of chitosan with or without other antifungal drugs on C. albicans and C. tropicalis. (3) Results: Treatment with chitosan in combination with fluconazole showed a great synergistic fungicidal effect against C. albicans and C. tropicalis, but an indifferent effect on antifungal activity when challenged with chitosan-amphotericin B or chitosan-caspofungin simultaneously. Furthermore, the combination of chitosan and fluconazole was effective against drug-resistant strains. (4) Conclusions: These findings provide strong evidence that chitosan in combination with fluconazole is a promising therapy against two Candida species and its drug-resistant strains.

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Essential gene analysis in Acinetobacter baumannii by high-density transposon mutagenesis and CRISPR interference

10.1101/2020.09.15.299016 ◽

2020 ◽

Author(s):

Jinna Bai ◽

Yunfei Dai ◽

Andrew Farinha ◽

Amy Y. Tang ◽

Sapna Syal ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Large Scale ◽

Essential Gene ◽

Essential Genes ◽

Require Time ◽

Crispr Interference ◽

Genetic Manipulations ◽

Multiple Processes ◽

Life Threatening ◽

Hospital Acquired

AbstractAcinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Developing rationally-designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii and we developed a CRISPR-interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC-family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii.ImportanceNew approaches are urgently needed to control A. baumannii, one of the most drug resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene-regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

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A Novel Method for Identifying Essential Genes by Fusing Dynamic Protein–Protein Interactive Networks

Genes ◽

10.3390/genes10010031 ◽

2019 ◽

Vol 10 (1) ◽

pp. 31 ◽

Cited By ~ 7

Author(s):

Fengyu Zhang ◽

Wei Peng ◽

Yunfei Yang ◽

Wei Dai ◽

Junrong Song

Keyword(s):

Drug Targets ◽

Essential Gene ◽

Gene Prediction ◽

Dynamic Networks ◽

Underlying Mechanism ◽

Essential Genes ◽

Data Sets ◽

Ppi Networks ◽

Yeast Data ◽

Novel Method

Essential genes play an indispensable role in supporting the life of an organism. Identification of essential genes helps us to understand the underlying mechanism of cell life. The essential genes of bacteria are potential drug targets of some diseases genes. Recently, several computational methods have been proposed to detect essential genes based on the static protein–protein interactive (PPI) networks. However, these methods have ignored the fact that essential genes play essential roles under certain conditions. In this work, a novel method was proposed for the identification of essential proteins by fusing the dynamic PPI networks of different time points (called by FDP). Firstly, the active PPI networks of each time point were constructed and then they were fused into a final network according to the networks’ similarities. Finally, a novel centrality method was designed to assign each gene in the final network a ranking score, whilst considering its orthologous property and its global and local topological properties in the network. This model was applied on two different yeast data sets. The results showed that the FDP achieved a better performance in essential gene prediction as compared to other existing methods that are based on the static PPI network or that are based on dynamic networks.

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Identification of Essential Genes in the Human Fungal Pathogen Aspergillus fumigatus by Transposon Mutagenesis

Eukaryotic Cell ◽

10.1128/ec.2.2.247-255.2003 ◽

2003 ◽

Vol 2 (2) ◽

pp. 247-255 ◽

Cited By ~ 48

Author(s):

Arnaud Firon ◽

François Villalba ◽

Roland Beffa ◽

Christophe d'Enfert

Keyword(s):

Aspergillus Fumigatus ◽

Transposable Element ◽

Drug Targets ◽

Fungal Infections ◽

Fungal Growth ◽

Developed Countries ◽

Transposon Mutagenesis ◽

Essential Genes ◽

Antifungal Drugs

ABSTRACT The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.

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Antifungal activity of MAF-1A peptide against Candida albicans

International Microbiology ◽

10.1007/s10123-021-00159-z ◽

2021 ◽

Author(s):

Rong Cheng ◽

Qiang Xu ◽

Fangfang Hu ◽

Hongling Li ◽

Bin Yang ◽

...

Keyword(s):

Candida Albicans ◽

Cell Membrane ◽

Antifungal Drugs ◽

Drug Resistant ◽

Fluorescent Staining ◽

Qrt Pcr ◽

Major Threat ◽

Antifungal Action ◽

Resistant Strains ◽

Drug Resistant Strains

AbstractInvasive candidiasis is a major threat to human health, and Candida albicans is the most common pathogenic species responsible for this condition. The incidence of drug-resistant strains of C. albicans is rising, necessitating the development of new antifungal drugs. Antimicrobial peptides (AMPs) have recently attracted attention due to their unique ability to evade the drug resistance of microorganisms. However, the mechanism of their activity has not yet been identified. The current study analyzed the mode of action of MAF-1A by confocal microscopy, scanning electron microscopy, fluorescent staining, flow cytometry, and qRT-PCR. The results indicate that MAF-1A disrupts the cell membrane of C. albicans and enters the cell where it binds and interacts with nucleic acids. qRT-PCR demonstrated that the expression of several sterol biosynthesis–related genes in C. albicans was increased after MAF-1A treatment. Together, these findings suggest that MAF-1A exerts antifungal action by affecting both the cell membrane and intracellular components. The antifungal mechanism of MAF-1A is unique, and its identification has great research and clinical significance.

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Increase of Virulence and Its Phenotypic Traits in Drug-Resistant Strains of Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01223-07 ◽

2008 ◽

Vol 52 (3) ◽

pp. 927-936 ◽

Cited By ~ 39

Author(s):

Letizia Angiolella ◽

Anna Rita Stringaro ◽

Flavia De Bernardis ◽

Brunella Posteraro ◽

Mariantonietta Bonito ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Biological Properties ◽

Antifungal Drugs ◽

Infection Model ◽

Phenotypic Traits ◽

Homozygous Mutation ◽

Drug Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

ABSTRACT There is concern about the rise of antifungal drug resistance, but little is known about comparative biological properties and pathogenicity of drug-resistant strains. We generated fluconazole (FLC; CO23RFLC)- or micafungin (FK; CO23RFK)-resistant strains of Candida albicans by treating a FLC- and FK-susceptible strain of this fungus (CO23S) with stepwise-increasing concentrations of either drug. Molecular analyses showed that CO23RFLC had acquired markedly increased expression of the drug-resistance efflux pump encoded by the MDR1 gene, whereas CO23RFK had a homozygous mutation in the FSK1 gene. These genetic modifications did not alter to any extent the growth capacity of the drug-resistant strains in vitro, either at 28°C or at 37°C, but markedly increased their experimental pathogenicity in a systemic mouse infection model, as assessed by the overall mortality and target organ invasion. Interestingly, no apparent increase in the vaginopathic potential of the strains was observed with an estrogen-dependent rat vaginal infection. The increased pathogenicity of drug-resistant strains for systemic infection was associated with a number of biochemical and physiological changes, including (i) marked cellular alterations associated with a different expression and content of major cell wall polysaccharides, (ii) more rapid and extensive hypha formation in both liquid and solid media, and (iii) increased adherence to plastic and a propensity for biofilm formation. Overall, our data demonstrate that experimentally induced resistance to antifungal drugs, irrespective of drug family, can substantially divert C. albicans biology, affecting in particular biological properties of potential relevance for deep-seated candidiasis.

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Visible Lights Combined with Photosensitizing Compounds Are Effective against Candida albicans Biofilms

Microorganisms ◽

10.3390/microorganisms9030500 ◽

2021 ◽

Vol 9 (3) ◽

pp. 500 ◽

Cited By ~ 1

Author(s):

Priyanka Bapat ◽

Gurbinder Singh ◽

Clarissa J. Nobile

Keyword(s):

Candida Albicans ◽

Photodynamic Therapy ◽

Biofilm Formation ◽

Fungal Pathogens ◽

Fungal Infections ◽

Therapeutic Strategies ◽

Antifungal Drugs ◽

Clinical Settings ◽

Drug Resistant ◽

Candida Albicans Biofilms

Fungal infections are increasing in prevalence worldwide, especially in immunocompromised individuals. Given the emergence of drug-resistant fungi and the fact that there are only three major classes of antifungal drugs available to treat invasive fungal infections, there is a need to develop alternative therapeutic strategies effective against fungal infections. Candida albicans is a commensal of the human microbiota that is also one of the most common fungal pathogens isolated from clinical settings. C. albicans possesses several virulence traits that contribute to its pathogenicity, including the ability to form drug-resistant biofilms, which can make C. albicans infections particularly challenging to treat. Here, we explored red, green, and blue visible lights alone and in combination with common photosensitizing compounds for their efficacies at inhibiting and disrupting C. albicans biofilms. We found that blue light inhibited biofilm formation and disrupted mature biofilms on its own and that the addition of photosensitizing compounds improved its antibiofilm potential. Red and green lights, however, inhibited biofilm formation only in combination with photosensitizing compounds but had no effects on disrupting mature biofilms. Taken together, these results suggest that photodynamic therapy may be an effective non-drug treatment for fungal biofilm infections that is worthy of further exploration.

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The SUMO protease Ulp2 regulates genome stability and drug resistance in the human fungal pathogen Candida albicans

10.1101/2021.12.06.471441 ◽

2021 ◽

Author(s):

Marzia Rizzo ◽

Natthapon Soisangwan ◽

Jan Soetaert ◽

Samuel Vega-Estevez ◽

Anna Selmecki ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Fungal Pathogen ◽

Genome Instability ◽

Antifungal Drugs ◽

Antifungal Drug ◽

Genome Plasticity ◽

Sumo Protease ◽

Human Fungal Pathogen ◽

Life Threatening

AbstractStress-induced genome instability in microbial organisms is emerging as a critical regulatory mechanism for driving rapid and reversible adaption to drastic environmental changes. In Candida albicans, a human fungal pathogen that causes life-threatening infections, genome plasticity confers increased virulence and antifungal drug resistance. Discovering the mechanisms regulating C. albicans genome plasticity is a priority to understand how this and other microbial pathogens establish life-threatening infections and develop resistance to antifungal drugs. We identified the SUMO protease Ulp2 as a critical regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to hypersensitivity to genotoxic agents and increased genome instability. This increased genome diversity causes reduced fitness under standard laboratory growth conditions but enhances adaptation to stress, making ulp2Δ/Δ cells more likely to thrive in the presence of antifungal drugs. Whole-genome sequencing indicates that ulp2Δ/Δ cells counteract antifungal drug-induced stress by developing segmental aneuploidies of chromosome R and chromosome I. We demonstrate that intrachromosomal repetitive elements drive the formation of complex novel genotypes with adaptive power.

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The non-essentiality of essential genes suggests a loss-of-function therapeutic strategy for loss-of-function human diseases

10.1101/040568 ◽

2016 ◽

Cited By ~ 1

Author(s):

Piaopiao Chen ◽

Dandan Wang ◽

Han Chen ◽

Zhenzhen Zhou ◽

Xionglei He

Keyword(s):

Side Effects ◽

Large Scale ◽

Therapeutic Strategy ◽

Essential Gene ◽

Large Population ◽

Essential Genes ◽

Type I ◽

Type Ii ◽

Loss Of Function ◽

Function Type

Essential genes refer to those whose null mutation leads to lethality or sterility. We propose that the fatal effect of inactivating an essential gene can be attributed to either the loss of indispensable core cellular function (type I), or the gain of fatal side effects after losing dispensable periphery function (type II). In principle, inactivation of the type I essential genes can be rescued only by re-gain of the core functions, whereas inactivation of the type II essential genes could be rescued by a further loss of function of another gene to eliminate the otherwise fatal side effects. Because such loss-of-function rescuing mutations may occur spontaneously, type II essential genes may become non-essential in a few individuals of a large population. We tested this idea in the yeastSacchromyces cerevisiae. Large-scale whole genome sequencing of such essentiality-reversing mutants reveals 14 cases where inactivation of an essential gene is rescued by loss-of-function mutations on another gene. In particular, the essential gene encoding the enzyme adenylosuccinate lyase (ADSL) is shown to be type II, suggesting a loss-of-function therapeutic strategy for the human disorder ADSL deficiency. A proof-of-principle test of this strategy in the nematodeCaenorhabditis elegansshows promising results.

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