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

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
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.

scholarly journals The 5’ untranslated region of theEFG1transcript promotes its translation to regulate hyphal morphogenesis inCandida albicans

2018 ◽  
Author(s):  
Prashant R. Desai ◽  
Klaus Lengeler ◽  
Mario Kapitan ◽  
Silas Matthias Janßen ◽  
Paula Alepuz ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Regulatory Mechanisms ◽  
Untranslated Regions ◽  
Regulatory Sequence ◽  
Transcriptional Level ◽  
Human Fungal Pathogen ◽  
Hyphal Morphogenesis ◽  
Cellular 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 1170 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 theEFG1ORF 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 context of the nativeEFG1promoter. The results suggest 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. 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 virulence ofC. albicansdepends on the combination of transcriptional and translation regulatory mechanisms.

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

2014 ◽  
Vol 83 (2) ◽  
pp. 637-645 ◽  
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.

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

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
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.

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

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
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.

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

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.

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

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
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.

scholarly journals Reconfiguration of Transcriptional Control of Lysine Biosynthesis in Candida albicans Involves a Central Role for the Gcn4 Transcriptional Activator

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Yumnam Priyadarshini ◽  
Krishnamurthy Natarajan
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Biosynthetic Pathway ◽  
Transcriptional Regulator ◽  
Lysine Biosynthesis ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Starvation Conditions

ABSTRACT Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candida albicans. We show that although both Saccharomyces cerevisiae and C. albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C. albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S. cerevisiae pathway-specific regulator Lys14. Evolution of transcriptional control is essential for organisms to cope with diversification into a spectrum of environments, including environments with limited nutrients. Lysine biosynthesis in fungi occurs in eight enzymatic steps. In Saccharomyces cerevisiae, amino acid starvation elicits the induction of LYS gene expression, mediated by the master regulator Gcn4 and the pathway-specific transcriptional regulator Lys14. Here, we have shown that the activation of LYS gene expression in the human fungal pathogen Candida albicans is predominantly controlled by Gcn4 under amino acid starvation conditions. Multiple lines of study showed that the four C. albicans LYS14-like genes have no role in the regulation of lysine biosynthesis. Whereas Gcn4 is dispensable for the growth of S. cerevisiae under lysine deprivation conditions, it is an essential regulator required for the growth of C. albicans under these conditions, as gcn4 deletion caused lysine auxotrophy. Gcn4 is required for the induction of increased LYS2 and LYS9 mRNA but not for the induction of increased LYS4 mRNA. Under lysine or isoleucine-valine deprivation conditions, Gcn4 recruitment to LYS2 and LYS9 promoters was induced in C. albicans. Indeed, in contrast to the S. cerevisiae LYS gene promoters, all LYS gene promoters in C. albicans harbored a Gcn4 binding site but not all harbored the S. cerevisiae Lys14 binding site, indicating the evolutionary divergence of cis-regulatory motifs. Thus, the transcriptional rewiring of the lysine biosynthetic pathway in C. albicans involves not only neofunctionalization of the four LYS14-like genes but the attendant strengthening of control by Gcn4, indicating a coordinated response with a much broader scope for control of amino acid biosynthesis in this human pathogen. IMPORTANCE Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candida albicans. We show that although both Saccharomyces cerevisiae and C. albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C. albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S. cerevisiae pathway-specific regulator Lys14.

scholarly journals mSphere of Influence: Considering Complex Mutational Processes That Shape Microbial Virulence

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Matthew Zack Anderson
Keyword(s):  
Infectious Disease ◽  
Candida Albicans ◽  
Gene Flow ◽  
Fungal Pathogen ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Microbial Adaptation ◽  
Oropharyngeal Infection ◽  
Mutational Processes ◽  
Selection Of

ABSTRACT Matt Anderson works in the field of genetics and infectious disease, with a focus on the human fungal pathogen Candida albicans. In this mSphere of Influence article, he reflects on how two papers, “Gene Flow Contributes to Diversification of the Major Fungal Pathogen Candida albicans” (J. Ropars, C. Maufrais, D. Diogo, M. Marcet-Houben, A. Perin, et al., Nat Commun 9:2253, 2018, https://doi.org/10.1038/s41467-018-04787-4) and “Selection of Candida albicans Trisomy during Oropharyngeal Infection Results in a Commensal-Like Phenotype” (A. Forche, N. V. Solis, M. Swidergall, R. Thomas, A. Guyer, et al., PLoS Genet 15:e1008137, 2019, https://doi.org/10.1371/journal.pgen.1008137), made an impact on him by incorporating less commonly investigated mechanisms of genome evolution into the context of microbial adaptation.

scholarly journals SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Ben A. Evans ◽  
Douglas A. Bernstein
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Life Threatening ◽  
Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

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

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Helene Tournu ◽  
Arielle Butts ◽  
Glen E. Palmer
Keyword(s):  
Protein Function ◽  
Fungal Pathogen ◽  
Specific Activity ◽  
Expression Level ◽  
Biosynthetic Activity ◽  
Content Type ◽  
Reading Frame ◽  
Human Fungal Pathogen ◽  
Progressive Loss ◽  
Conditional Expression

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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