Genome-Wide Fitness Test and Mechanism-of-Action Studies of Inhibitory Compounds in Candida albicans

Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical–genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery

Essential Gene Identification and Drug Target Prioritization in Aspergillus fumigatus

Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BΔ, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors

Mechanism-of-Action Determination of GMP Synthase Inhibitors and Target Validation in Candida albicans and Aspergillus fumigatus

SummaryMechanism-of-action (MOA) studies of bioactive compounds are fundamental to drug discovery. However, in vitro studies alone may not recapitulate a compound's MOA in whole cells. Here, we apply a chemogenomics approach in Candida albicans to evaluate compounds affecting purine metabolism. They include the IMP dehydrogenase inhibitors mycophenolic acid and mizoribine and the previously reported GMP synthase inhibitors acivicin and 6-diazo-5-oxo-L-norleucine (DON). We report important aspects of their whole-cell activity, including their primary target, off-target activity, and drug metabolism. Further, we describe ECC1385, an inhibitor of GMP synthase, and provide biochemical and genetic evidence supporting its MOA to be distinct from acivicin or DON. Importantly, GMP synthase activity is conditionally essential in C. albicans and Aspergillus fumigatus and is required for virulence of both pathogens, thus constituting an unexpected antifungal target

Heterogeneity in cblG : differential binding of vitamin B12 to methionine synthase

Fibroblasts from patients with functional methionine synthase deficiency can be divided into 2 complementation classes, cblE and cblG. Both have low levels of intracellular methylcobalamin. Both groups also demonstrate low levels of incorporation of label from 5-methyltetrahydrofolate into macromolecules. Under standard reducing conditions, methionine synthase specific activity is normal in cblE fibroblast extracts, but is low in cblG fibroblast extracts. Seven cblE and seven out of ten cblG cell lines demonstrate levels of accumulation of ($sp{57}$Co) CN-Cbl in fibroblasts comparable to that of control cells. They exhibit similar proportions of label associated with the two intracellular cobalamin binders, methylmalonyl-CoA mutase and methionine synthase. The remaining three cblG cell lines exhibit a lower level of cobalamin accumulation, and demonstrate a lack of cobalamin association with the enzyme methionine synthase. The specific activity of methionine synthase is almost undetectable in the three cblG cell lines that showed no such association. These results demonstrate heterogeneity within the cblG group and suggest that the defect in cblG affects the methionine synthase apoenzyme

Expressional Level Analysis of p<i>NiiA</i>-CPR Mutants

<div><p>(A) RT-PCR of p<i>NiiA-MET2:</i> (a) p<i>NiiA</i>-<i>MET2</i> mutant under inducing conditions, (b) p<i>NiiA</i>-<i>MET2</i> mutant under repressing conditions plus 100 μg/ml methionine, (c) p<i>NiiA</i>-<i>MET2</i> mutant under inducing condition plus 100 μg/ml methionine, and (d) wild-type A. fumigatus strain (CEA10) under repressing conditions. To monitor and ensure even sample loading, RT-PCRs for the <i>ACT1</i> transcript were also performed using identical samples. In addition, standard PCR was performed to confirm that there is no detectable genomic-DNA contamination.</p><p>(B) Northern blot analysis of p<i>NiiA</i>-<i>ALR1</i> mutant expression levels. Northern blot was performed with RNA samples prepared from the nonessential <i>ALR1</i> mutant and wild-type cells growing in standard inducing (In) or repressing (Re) conditions. <i>ACT1</i> transcript levels served as sample loading controls.</p><p>(C) Real-time RT-PCR analysis of expression level of p<i>NiiA</i>-<i>ALR1</i> and p<i>NiiA</i>-<i>MET2</i>. p<i>NiiA</i>-CPR mutants and wild-type strain (CEA10) were grown in inducing (In) or repressing (Re) medium at 37 °C for 20 h, and total RNA was extracted from identical time points. The relative expression level normalized to total input RNA [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-b043" target="_blank">43</a>] is displayed on the <i>y</i>-axis. Error bars represent SD. Compared to wild-type, the relative expression level for <i>ALR1</i> and <i>MET2</i> is 0.013 and 0.061 under repressing conditions and 2.49 and 23.58 under inducing conditions, respectively.</p><p>(D) <i>pNiiA-TUB1</i> expression level under inducing (In) and repressing (Re) conditions versus wild-type level is displayed on the <i>y</i>-axis. Error bar represents SD. The relative expression level of p<i>NiiA-TUB1</i> versus wild-type is 0.06 under repressing conditions and 6.35 under inducing conditions, respectively. Note: Since real-time RT-PCR was performed using primers detecting both p<i>NiiA-TUB1</i> and wild-type allele, data shown were calculated by subtracting wild-type level from total inducing (In) and total repressing (Re) level, respectively.</p></div

In Vivo Validation of A. fumigatus p<i>NiiA</i>-CPR Mutants in an Immunocompromised Murine Model of Systemic Infection

<div><p>(A) p<i>NiiA</i>-CPR mutants growing on AMM plus 20% mouse serum (Sigma) reproduced their terminal growth phenotypes as observed on either AMM plus ammonium or rich medium (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-t002" target="_blank">Table 2</a>).</p><p>(B and C) In vivo validation of p<i>NiiA</i>-CPR mutants. (B) ICR male mice were immunocompromised by administration of cyclophosphamide at 150 mg/kg twice prior to infection and 100 mg/kg twice a week after infection. Approximately 10⁵ viable conidia from individual p<i>NiiA</i>-CPR mutants <i>(TUB1, SEC31, GCD6, GFA1, MET2,</i> and <i>AUR1)</i> were injected into the tail vein of immunocompromised mice (five mice per group). Signs of infection were monitored for up to 12 d following infection. Wild-type strain CEA10 and the starting strain CEA17 (a <i>pyrG</i>⁻ auxotroph of CEA10) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-b025" target="_blank">25</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-b039" target="_blank">39</a>] were included as positive controls for virulence and avirulence, respectively.</p><p>(C) Genetic inactivation of the <i>ERG11</i> gene family promotes avirulence in an immunocompromised murine model of systemic infection. Pathogenesis of <i>erg11A</i>Δ, <i>erg11B</i>Δ, and an <i>ERG11</i> double mutant <i>(erg11B</i>Δ, p<i>NiiA-ERG11A)</i> was similarly analyzed (as described above) but over a longer postinfection period (22 d), and animal survival was compared to CEA10 and CEA17 control strains.</p></div

Determination of Cidal or Static Terminal Phenotypes

<div><p>A representative example of cidal and static terminal phenotypes for <i>GFA1</i> and <i>TRR1</i> p<i>NiiA</i>-CPR mutants is shown.</p><p>(A) p<i>NiiA</i>-GFA1 displayed a cidal terminal phenotype as a dramatic (greater than 90%) reduction in CFU was observed after incubation in p<i>NiiA</i>-repressing conditions for 24 or 48 h.</p><p>(B) p<i>NiiA</i>-<i>TRR1</i> revealed a static terminal phenotype as no significant reduction in CFU counts was detected after 48-h incubation under repressing conditions. A summary of all additional cidal/static terminal phenotypes for A. fumigatus genes displaying 4+ qualitative growth phenotypes is provided (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-t002" target="_blank">Table 2</a>).</p></div

Phenotypic Analyses of <i>ERG11</i> Gene Family

<p>Growth phenotypes of <i>ERG11</i> gene family were observed with p<i>NiiA</i>-CPR mutants, null deletion mutants, and double mutants <i>(erg11B</i>Δ, p<i>NiiA-ERG11A).</i> Strains were grown on either inducing medium (AMM plus nitrate) or repressing medium (AMM plus ammonium) at 30 °C for 40 h.</p

Analysis of p<i>NiiA</i>-CPR Associated Morphological Terminal Phenotypes

<p>Terminal growth phenotypes of p<i>NiiA</i>-CPR mutants were observed under a microscope (×160) with conidia grown for 36 to 40 h at 30 °C under standard repressing conditions. A continuum of conidia germination phenotypes of high penetrance was observed; ranging from those completely failing to undergo polarized growth <i>(SEC31, SLY1)</i> or swollen and highly disorganized condidia <i>(GFA1),</i> to those displaying stunted <i>(TUB1, ERG10)</i> or nonbranching germlings with swollen conidia <i>(HEM15)</i> with only rudimentary polarized growth. Micromycelial colonies were observed for a p<i>NiiA</i>-<i>FKS1</i> mutant and resembling the morphology of wild-type A. fumigatus when grown in the presence of minimum effective concentration (MEC) of the FKS1p inhibitor, caspofungin [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-b021" target="_blank">21</a>]. Growth phenotypes under inducing conditions are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-sg003" target="_blank">Figure S3</a>.</p