Titrating Gene Function in the Human Fungal PathogenCandida albicansthrough Poly-Adenosine Tract Insertion

ABSTRACTA recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein’s expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen,Candida albicans. Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of theC. albicanslanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted andC. albicanssusceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series ofC. albicansstrains with 3 to 12 AAA codons inserted at the 5′ end of theARO1gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate ofC. albicansin standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function inC. albicans.IMPORTANCEInvestigating a protein’s functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach—the insertion of poly-A repeats within the coding sequence of the gene—is sufficient to modulate its function in the prevalent human fungal pathogenC. albicans. Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.

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The 5′ Untranslated Region of theEFG1Transcript Promotes Its Translation To Regulate Hyphal Morphogenesis inCandida albicans

mSphere ◽

10.1128/msphere.00280-18 ◽

2018 ◽

Vol 3 (4) ◽

Cited By ~ 2

Author(s):

Prashant R. Desai ◽

Klaus Lengeler ◽

Mario Kapitan ◽

Silas Matthias Janßen ◽

Paula Alepuz ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Untranslated Regions ◽

Regulatory Sequence ◽

Transcriptional Level ◽

Content Type ◽

Reading Frame ◽

Human Fungal Pathogen ◽

Hyphal Morphogenesis ◽

Incandida Albicans

ABSTRACTExtensive 5′ untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis inCandida albicans. The major transcripts of theEFG1gene, which are responsible for cellular morphogenesis and metabolism, contain a 5′ UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5′ UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of theEFG1transcript. Polysomal analyses revealed that the 218-nt 5′ UTR sequence is required for efficient translation of the Efg1 protein. Replacement of theEFG1open reading frame (ORF) by the heterologous reporter geneCaCBGlucconfirmed the positive regulatory importance of the identified 5′ UTR sequence. In contrast to other reported transcripts containing extensive 5′ UTR sequences, these results indicate the positive translational function of the 5′ UTR sequence in theEFG1transcript, which is observed in the context of the nativeEFG1promoter. It is proposed that the 5′ UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of theEFG1transcript.IMPORTANCEMany of the virulence traits that makeCandida albicansan important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5′ untranslated regulatory sequence (5′ UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5′ UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5′ UTR sequences, it appears that the virulence ofC. albicansdepends on the combination of transcriptional and translational regulatory mechanisms.

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mSphere of Influence: the Power of Yeast Genetics Still Going Strong!

mSphere ◽

10.1128/msphere.00647-19 ◽

2019 ◽

Vol 4 (5) ◽

Author(s):

Felipe H. Santiago-Tirado

Keyword(s):

Genetic Analysis ◽

Fungal Pathogen ◽

Cell Biology ◽

Genetic Manipulation ◽

Chemical Genetics ◽

Yeast Genetics ◽

Content Type ◽

Human Fungal Pathogen ◽

Link Type ◽

Fungal Meningitis

ABSTRACT Felipe Santiago-Tirado studies the cell biology of cryptococcal infections. In this mSphere of Influence article, he reflects on how the papers “Systematic Genetic Analysis of Virulence in the Human Fungal Pathogen Cryptococcus neoformans” (https://doi.org/10.1016/j.cell.2008.07.046) and “Unraveling the Biology of a Fungal Meningitis Pathogen Using Chemical Genetics” (https://doi.org/10.1016/j.cell.2014.10.044) by the Noble and Madhani groups influenced his thinking by showcasing the various modern applications of yeast genetics in an organism where genetic manipulation was difficult.

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Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis

mSphere ◽

10.1128/msphere.00084-15 ◽

2016 ◽

Vol 1 (2) ◽

Cited By ~ 16

Author(s):

Shannon K. Esher ◽

Kyla S. Ost ◽

Lukasz Kozubowski ◽

Dong-Hoon Yang ◽

Min Su Kim ◽

...

Keyword(s):

Subcellular Localization ◽

Cryptococcus Neoformans ◽

Posttranslational Modification ◽

Fungal Pathogen ◽

Content Type ◽

Processing Enzymes ◽

Human Fungal Pathogen ◽

Modification Process ◽

Substrate Proteins ◽

Processing Steps

ABSTRACT Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus. Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes through a posttranslational modification process known as prenylation. These studies investigate the roles of one of the prenylation enzymes, farnesyltransferase, as well as the postprenylation processing enzymes in C. neoformans. We demonstrate that the postprenylation processing steps are dispensable for the localization of certain substrate proteins. However, both protein farnesylation and the subsequent postprenylation processing steps are required for full pathogenesis of this fungus.

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Protection from Systemic Candida albicans Infection by Inactivation of the Sts Phosphatases

Infection and Immunity ◽

10.1128/iai.02789-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 637-645 ◽

Cited By ~ 19

Author(s):

Shamoon Naseem ◽

David Frank ◽

James B. Konopka ◽

Nick Carpino

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Fungal Growth ◽

Systemic Infection ◽

Content Type ◽

Host Immune Responses ◽

Human Fungal Pathogen ◽

Rapid Induction ◽

Inflammatory Damage ◽

Functional Inactivation

The human fungal pathogenCandida albicanscauses invasive candidiasis, characterized by fatal organ failure due to disseminated fungal growth and inflammatory damage. Thesuppressor ofTCRsignaling 1 (Sts-1) and Sts-2 are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic cell lineages, including T lymphocytes, mast cells, and platelets. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection byC. albicans, such that greater than 80% of mice lacking Sts-1 and -2 survive a dose ofC. albicans(2.5 × 105CFU/mouse) that is uniformly lethal to wild-type mice within 10 days. Restriction of fungal growth within the kidney occurs by 24 h postinfection in the mutant mice. This occurs without induction of a hyperinflammatory response, as evidenced by the decreased presence of leukocytes and inflammatory cytokines that normally accompany the antifungal immune response. Instead, the absence of the Sts phosphatases leads to the rapid induction of a unique immunological environment within the kidney, as indicated by the early induction of a proinflammatory cytokine (CXL10). Mice lacking either Sts enzyme individually display an intermediate lethality phenotype. These observations identify an opportunity to optimize host immune responses toward a deadly fungal pathogen.

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Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

mSphere ◽

10.1128/msphere.00385-16 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Henry Ng ◽

Neta Dean

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Virulence Factors ◽

Fungal Pathogen ◽

Fungal Virulence ◽

Important Conclusion ◽

Content Type ◽

Guide Rna ◽

Improve Efficiency ◽

Human Fungal Pathogen

ABSTRACT Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans. The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

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Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

mSphere ◽

10.1128/msphere.00379-17 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 3

Author(s):

Arielle Butts ◽

Christian DeJarnette ◽

Tracy L. Peters ◽

Josie E. Parker ◽

Morgan E. Kerns ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Second Generation ◽

Active Compounds ◽

Whole Cell ◽

Competitive Fitness ◽

Content Type ◽

Human Fungal Pathogen ◽

Conventional Drug ◽

Physiologically Active Compounds

ABSTRACT Conventional drug screening typically employs either target-based or cell-based approaches. The first group rely on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound. Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.

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Antimicrobial Octapeptin C4 Analogues Active against Cryptococcus Species

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00986-17 ◽

2017 ◽

Vol 62 (2) ◽

Cited By ~ 2

Author(s):

Jessica L. Chitty ◽

Mark S. Butler ◽

Azzah Suboh ◽

David J. Edwards ◽

Matthew A. Cooper ◽

...

Keyword(s):

Fungal Pathogen ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Content Type ◽

Human Fungal Pathogen ◽

Cyclic Lipopeptide ◽

Structure Activity ◽

Cryptococcus Species ◽

Insight Into ◽

Better Than

ABSTRACTResistance to antimicrobials is a growing problem in both developed and developing countries. In nations where AIDS is most prevalent, the human fungal pathogenCryptococcus neoformansis a significant contributor to mortality, and its growing resistance to current antifungals is an ever-expanding threat. We investigated octapeptin C4, from the cationic cyclic lipopeptide class of antimicrobials, as a potential new antifungal. Octapeptin C4 was a potent, selective inhibitor of this fungal pathogen with an MIC of 1.56 μg/ml. Further testing of octapeptin C4 against 40 clinical isolates ofC. neoformansvar.grubiiorneoformansshowed an MIC of 1.56 to 3.13 μg/ml, while 20 clinical isolates ofC. neoformansvar.gattiihad an MIC of 0.78 to 12.5 μg/ml. In each case, the MIC values for octapeptin C4 were equivalent to, or better than, current antifungal drugs fluconazole and amphotericin B. The negatively charged polysaccharide capsule ofC. neoformansinfluences the pathogen's sensitivity to octapeptin C4, whereas the degree of melanization had little effect. Testing synthetic octapeptin C4 derivatives provided insight into the structure activity relationships, revealing that the lipophilic amino acid moieties are more important to the activity than the cationic diaminobutyric acid groups. Octapeptins have promising potential for development as anticryptococcal therapeutic agents.

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Troponoids Can Inhibit Growth of the Human Fungal Pathogen Cryptococcus neoformans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02574-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 15

Author(s):

Maureen J. Donlin ◽

Anthony Zunica ◽

Ashlyn Lipnicky ◽

Aswin K. Garimallaprabhakaran ◽

Alex J. Berkowitz ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Amphotericin B ◽

Fungal Pathogen ◽

Antimicrobial Activities ◽

Fungal Species ◽

Content Type ◽

Human Fungal Pathogen ◽

Red Cedar ◽

Immunosuppressed Patients ◽

Western Red Cedar

ABSTRACT Cryptococcus neoformans is a pathogen that is common in immunosuppressed patients. It can be treated with amphotericin B and fluconazole, but the mortality rate remains 15 to 30%. Thus, novel and more effective anticryptococcal therapies are needed. The troponoids are based on natural products isolated from western red cedar, and have a broad range of antimicrobial activities. Extracts of western red cedar inhibit the growth of several fungal species, but neither western red cedar extracts nor troponoid derivatives have been tested against C. neoformans. We screened 56 troponoids for their ability to inhibit C. neoformans growth and to assess whether they may be attractive candidates for development into anticryptococcal drugs. We determined MICs at which the compounds inhibited 80% of cryptococcal growth relative to vehicle-treated controls and identified 12 compounds with MICs ranging from 0.2 to 15 μM. We screened compounds with MICs of ≤20 μM for cytotoxicity in liver hepatoma cells. Fifty percent cytotoxicity values (CC50s) ranged from 4 to >100 μM. The therapeutic indexes (TI, CC50/MIC) for most of the troponoids were fairly low, with most being <8. However, two compounds had TI values that were >8, including a tropone with a TI of >300. These tropones are fungicidal and are not antagonistic when used in combination with fluconazole or amphotericin B. Inhibition by these two tropones remains unchanged under conditions favoring cryptococcal capsule formation. These data support the hypothesis that troponoids may be a productive scaffold for the development of novel anticryptococcal therapies.

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Aneuploidy Underlies Tolerance and Cross-Tolerance to Drugs in Candida parapsilosis

Microbiology Spectrum ◽

10.1128/spectrum.00508-21 ◽

2021 ◽

Author(s):

Feng Yang ◽

Hui Lu ◽

Hao Wu ◽

Ting Fang ◽

Judith Berman ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Candida Parapsilosis ◽

Cross Tolerance ◽

Content Type ◽

Human Fungal Pathogen

Candida parapsilosis is an emerging major human fungal pathogen, especially in neonates. Aneuploidy, having uneven numbers of chromosomes, is a well-known mechanism for adapting to stress in Candida albicans , the most common human fungal pathogen.

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Candida albicans CUG Mistranslation Is a Mechanism To Create Cell Surface Variation

mBio ◽

10.1128/mbio.00285-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 51

Author(s):

Isabel Miranda ◽

Ana Silva-Dias ◽

Rita Rocha ◽

Rita Teixeira-Santos ◽

Carolina Coelho ◽

...

Keyword(s):

Candida Albicans ◽

Cell Surface ◽

Fungal Pathogen ◽

Single Gene ◽

Yeast Cells ◽

Phenotypic Switching ◽

Leucine Incorporation ◽

Host Immune System ◽

Content Type ◽

Human Fungal Pathogen

ABSTRACT In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface β-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions. IMPORTANCE The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule β-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution.

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