Identification of genome-wide alternative splicing events in sequential, isogenic clinical isolates of Candida albicans reveals a mechanism important for drug resistance and tolerance to cellular stress

Alternative gene splicing (AS) is a process by which a single gene can give rise to different protein isoforms, generating proteome diversity. Despite recent advances in our understanding of AS in basic cellular processes, the role of AS in drug resistance and fungal pathogenesis is poorly understood. In Candida albicans, approximately 6% of the genes contain introns. Considering this low and random distribution of introns, we focused our study on alternative splicing (AS) and its impact on the development of drug resistance, an area largely unexplored in this yeast. We performed comparative RNA sequencing of sequential isogenic azole sensitive and resistant isolates of C. albicans. The analysis revealed differential expression of splice junctions/isoforms in 14 genes, between the drug sensitive and resistant isolates. Furthermore, C. albicans WT cells exposed to antifungal drugs, heat stress or metal deficiency also showed differential expression of isoforms for the genes undergoing AS. In this study we present data on the effect of AS on the function of SOD3. The C. albicans SOD3 has a single intron and is important for the removal of superoxide radicals. The overexpression of the two isoforms of SOD3 in its null background highlighted importance of spliced isoform in complementing the susceptibility to menadione. However, the two isoforms did not differ in rescuing the susceptibility of sod3Δ/Δto Amphotericin B. Collectively, these data suggest that AS may be a novel mechanism in C. albicans for stress adaptation and overcoming drug resistance.

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Identification of Genomewide Alternative Splicing Events in Sequential, Isogenic Clinical Isolates of Candida albicans Reveals a Novel Mechanism of Drug Resistance and Tolerance to Cellular Stresses

mSphere ◽

10.1128/msphere.00608-20 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 1

Author(s):

Suraya Muzafar ◽

Ravi Datta Sharma ◽

Abdul Haseeb Shah ◽

Naseem A. Gaur ◽

Ujjaini Dasgupta ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Alternative Splicing ◽

Rna Sequencing ◽

Single Gene ◽

Drug Susceptibility ◽

Protein Isoforms ◽

Sequencing Data ◽

Splice Isoforms ◽

Content Type

ABSTRACT Alternative splicing (AS)—a process by which a single gene gives rise to different protein isoforms in eukaryotes—has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans, has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans. Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3, which contains a single intron. The sod3Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicans. IMPORTANCE The emergence of resistance in Candida albicans, an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans, here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4505-4512.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4505-4512 ◽

Cited By ~ 66

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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DoChaP: The Domain Change Presenter

10.1101/2020.12.16.423045 ◽

2020 ◽

Author(s):

Shani T. Gal-Oz ◽

Nimrod Haiat ◽

Dana Eliyahu ◽

Guy Shani ◽

Tal Shay

Keyword(s):

Alternative Splicing ◽

Rna Splicing ◽

Protein Domains ◽

Visual Presentation ◽

Structural Effect ◽

Protein Isoforms ◽

Multiple Transcripts ◽

Genome Wide ◽

Health And Disease ◽

Specific Alternative

AbstractAlternative RNA splicing results in multiple transcripts of the same gene, possibly encoding for different protein isoforms with different protein domains and functionalities. Whereas it is possible to manually determine the effect of a specific alternative splicing event on the domain composition of a particular encoded protein, the process requires the tedious integration of several data sources; it is therefore error prone and its implementation is not feasible for genome-wide characterization of domains affected by differential splicing. To fulfill the need for an automated solution, we developed the Domain Change Presenter (DoChaP), a web server for the visualization of the exon–domain association. DoChaP visualizes all transcripts of a given gene, the domains of the proteins that they encode, and the exons encoding each domain. The visualization enables a comparison between the transcripts and between the protein isoforms they encode for. The organization and visual presentation of the information makes the structural effect of each alternative splicing event on the protein structure easily identified. To enable a study of the conservation of the exon structure, alternative splicing, and the effect of alternative splicing on protein domains, DoChaP also facilitates an inter-species comparison of domain–exon associations. DoChaP thus provides a unique and easy-to-use visualization of the exon–domain association and its conservation between transcripts and orthologous genes and will facilitate the study of the functional effects of alternative splicing in health and disease.

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Alternative splicing in endothelial cells: novel therapeutic opportunities in cancer angiogenesis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01753-1 ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Anna Di Matteo ◽

Elisa Belloni ◽

Davide Pradella ◽

Ambra Cappelletto ◽

Nina Volf ◽

...

Keyword(s):

Alternative Splicing ◽

Human Cancer ◽

Single Gene ◽

Predictive Biomarkers ◽

Protein Isoforms ◽

Therapeutic Interventions ◽

Cancer Therapies ◽

Functional Changes ◽

Splicing Regulators ◽

Multiple Protein

AbstractAlternative splicing (AS) is a pervasive molecular process generating multiple protein isoforms, from a single gene. It plays fundamental roles during development, differentiation and maintenance of tissue homeostasis, while aberrant AS is considered a hallmark of multiple diseases, including cancer. Cancer-restricted AS isoforms represent either predictive biomarkers for diagnosis/prognosis or targets for anti-cancer therapies. Here, we discuss the contribution of AS regulation in cancer angiogenesis, a complex process supporting disease development and progression. We consider AS programs acting in a specific and non-redundant manner to influence morphological and functional changes involved in cancer angiogenesis. In particular, we describe relevant AS variants or splicing regulators controlling either secreted or membrane-bound angiogenic factors, which may represent attractive targets for therapeutic interventions in human cancer.

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Effects of Azole Antifungal Drugs on the Transition from Yeast Cells to Hyphae in Susceptible and Resistant Isolates of the Pathogenic Yeast Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.4.763 ◽

1999 ◽

Vol 43 (4) ◽

pp. 763-768 ◽

Cited By ~ 52

Author(s):

Kien C. Ha ◽

Theodore C. White

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Molecular Mechanisms ◽

Opportunistic Infections ◽

Antifungal Drugs ◽

Yeast Cells ◽

Pathogenic Yeast ◽

Oral Infections ◽

Antifungal Drug Resistance ◽

Hyphal Formation

ABSTRACT Oral infections caused by the yeast Candida albicansare some of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug resistance, creating a major problem in the treatment of yeast infections in AIDS patients and other immunocompromised individuals. Several molecular mechanisms that contribute to drug resistance have been identified. InC. albicans, the ability to morphologically switch from yeast cells (blastospores) to filamentous forms (hyphae) is an important virulence factor which contributes to the dissemination ofCandida in host tissues and which promotes infection and invasion. A positive correlation between the level of antifungal drug resistance and the ability to form hyphae in the presence of azole drugs has been identified. Under hypha-inducing conditions in the presence of an azole drug, resistant clinical isolates form hyphae, while susceptible yeast isolates do not. This correlation is observed in a random sample from a population of susceptible and resistant isolates and is independent of the mechanisms of resistance.35S-methionine incorporation suggests that growth inhibition is not sufficient to explain the inhibition of hyphal formation, but it may contribute to this inhibition.

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The inositol polyphosphate kinase Ipk1 transcriptionally regulates mitochondrial functions in Candida albicans

FEMS Yeast Research ◽

10.1093/femsyr/foaa050 ◽

2020 ◽

Vol 20 (6) ◽

Author(s):

Hangqi Zhu ◽

Nali Zhu ◽

Liping Peng ◽

Bing Zhang ◽

Qilin Yu ◽

...

Keyword(s):

Candida Albicans ◽

Mitochondrial Function ◽

Antifungal Drugs ◽

Infection Model ◽

Polyphosphate Kinase ◽

Inositol Polyphosphate ◽

Human Beings ◽

Respiratory Chain Complexes ◽

Cellular Processes ◽

Mitochondrial Functions

ABSTRACT Inositol polyphosphates (IPs) is an important family of signaling molecules that regulate multiple cellular processes, such as chromatin remodeling, transcription and mRNA export. Inositol polyphosphate kinases, as the critical enzymes for production and transformation of IPs, directly determine the intracellular levels of IPs and therefore are involved in many cellular processes. However, its roles in Candida albicans, the leading fungal pathogen in human beings, remain to be investigated. In this study, we identified the inositol polyphosphate kinase Ipk1 in C. albicans and found that it localizes in the nucleus. Moreover, in the ipk1Δ/Δ mutant, the activity of mitochondrial respiratory chain complexes and the mitochondrial function was severely impaired, which were associated with down-regulation of mitochondrial function-related genes revealed by transcription profiling analysis. The ipk1Δ/Δ mutant also displayed hypersensitivity to a series of environmental stresses, such as antifungal drugs, oxidants, cell wall perturbing agents and macrophage attacks, followed by attenuation of virulence in a mouse systematic infection model. These findings firstly reported the importance of inositol polyphosphate kinase Ipk1 in C. albicans, especially its role in mitochondrial function maintenance and pathogenicity.

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Influence of Intron Length on Alternative Splicing of CD44

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5930 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5930-5941 ◽

Cited By ~ 49

Author(s):

Martyn V. Bell ◽

Alison E. Cowper ◽

Marie-Paule Lefranc ◽

John I. Bell ◽

Gavin R. Screaton

Keyword(s):

Alternative Splicing ◽

Genomic Structure ◽

Single Gene ◽

Intron Length ◽

Protein Isoforms ◽

Major Influence ◽

Eukaryotic Genes ◽

Alternatively Spliced ◽

Alternatively Spliced Exons

ABSTRACT Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

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Drug Resistance Is Not Directly Affected by Mating Type Locus Zygosity in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.4.1207-1212.2003 ◽

2003 ◽

Vol 47 (4) ◽

pp. 1207-1212 ◽

Cited By ~ 30

Author(s):

Claude Pujol ◽

Shawn A. Messer ◽

Michael Pfaller ◽

David R. Soll

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Mating Type ◽

Drug Susceptibility ◽

Antifungal Drugs ◽

Heterozygous Parent ◽

Type Locus ◽

Mating Type Locus ◽

Fluconazole Resistant ◽

Intrinsic Drug Resistance

ABSTRACT Recently, evidence was presented that in a collection of fluconazole-resistant strains of Candida albicans there was a much higher proportion of homozygotes for the mating type locus (MTL) than in a collection of fluconazole-sensitive isolates, suggesting the possibility that when cells become MTL homozygous they acquire intrinsic drug resistance. To investigate this possibility, an opposite strategy was employed. First, drug susceptibility was measured in a collection of isolates selected for MTL homozygosity. The majority of these isolates had not been exposed to antifungal drugs. Second, the level of drug susceptibility was compared between spontaneously generated MTL-homozygous progeny and their MTL-heterozygous parent strains which had not been exposed to antifungal drugs. The results demonstrate that naturally occurring MTL-homozygous strains are not intrinsically more drug resistant, supporting the hypotheses that either the higher incidence of MTL homozygosity previously demonstrated among fluconazole-resistant isolates involved associated homozygosity of a drug resistance gene linked to the MTL locus, or that MTL-homozygous strains may be better at developing drug resistance upon exposure to the drug than MTL-heterozygous strains. Furthermore, the results demonstrate that a switch by an MTL-homozygous strain from the white to opaque phenotype, the latter functioning as the facilitator of mating, does not notably alter drug susceptibility.

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Genome-wide Mapping of the Coactivator Ada2p Yields Insight into the Functional Roles of SAGA/ADA Complex inCandida albicans

Molecular Biology of the Cell ◽

10.1091/mbc.e08-11-1093 ◽

2009 ◽

Vol 20 (9) ◽

pp. 2389-2400 ◽

Cited By ~ 51

Author(s):

Adnane Sellam ◽

Christopher Askew ◽

Elias Epp ◽

Hugo Lavoie ◽

Malcolm Whiteway ◽

...

Keyword(s):

Drug Resistance ◽

Resistance Genes ◽

Target Genes ◽

Transferase Activity ◽

Histone Acetyl Transferase ◽

Functional Roles ◽

Cellular Processes ◽

Oxidative Resistance ◽

Genome Wide ◽

Insight Into

The SAGA/ADA coactivator complex, which regulates numerous cellular processes by coordinating histone acetylation, is widely conserved throughout eukaryotes, and analysis of the Candida albicans genome identifies the components of this complex in the fungal pathogen. We investigated the multiple functions of SAGA/ADA in C. albicans by determining the genome-wide occupancy of Ada2p using chromatin immunoprecipitation (ChIP). Ada2p is recruited to 200 promoters upstream of genes involved in different stress-response functions and metabolic processes. Phenotypic and transcriptomic analysis of ada2 mutant showed that Ada2p is required for the responses to oxidative stress, as well as to treatments with tunicamycin and fluconazole. Ada2p recruitment to the promoters of oxidative resistance genes is mediated by the transcription factor Cap1p, and coactivator function were also established for Gal4p, which recruits Ada2p to the promoters of glycolysis and pyruvate metabolism genes. Cooccupancy of Ada2p and the drug resistance regulator Mrr1p on the promoters of core resistance genes characterizing drug resistance in clinical strains was also demonstrated. Ada2p recruitment to the promoters of these genes were shown to be completely dependent on Mrr1p. Furthermore, ADA2 deletion causes a decrease in H3K9 acetylation levels of target genes, thus illustrating its importance for histone acetyl transferase activity.

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Splicing Regulators and Their Roles in Cancer Biology and Therapy

BioMed Research International ◽

10.1155/2015/150514 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 9

Author(s):

Maria Roméria da Silva ◽

Gabriela Alves Moreira ◽

Ronni Anderson Gonçalves da Silva ◽

Éverton de Almeida Alves Barbosa ◽

Raoni Pais Siqueira ◽

...

Keyword(s):

Alternative Splicing ◽

Cancer Biology ◽

Single Gene ◽

Tumor Biology ◽

Sr Proteins ◽

Protein Isoforms ◽

Protein Expression Pattern ◽

Regulatory Factors ◽

Impact Pathways ◽

Splicing Regulators

Alternative splicing allows cells to expand the encoding potential of their genomes. In this elegant mechanism, a single gene can yield protein isoforms with even antagonistic functions depending on the cellular physiological context. Alterations in splicing regulatory factors activity in cancer cells, however, can generate an abnormal protein expression pattern that promotes growth, survival, and other processes, which are relevant to tumor biology. In this review, we discuss dysregulated alternative splicing events and regulatory factors that impact pathways related to cancer. The SR proteins and their regulatory kinases SRPKs and CLKs have been frequently found altered in tumors and are examined in more detail. Finally, perspectives that support splicing machinery as target for the development of novel anticancer therapies are discussed.

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