Potential Target For Antifungal Drugs Research Articles

Biosynthesis of iron-chelating terramides A-C and their role in Aspergillus terreus infection

Fungal natural products from various species often feature hydroxamic acid motifs that have the ability to chelate iron. These compounds have an array of medicinally and ecologically relevant activities. Through genome mining, gene deletion in the host Aspergillus terreus, and heterologous expression experiments, this study has revealed that a nonribosomal peptide synthetase (NRPS) TamA and a specialized cytochrome P450 monooxygenase TamB catalyze the sequential biosynthetic reactions in the formation of terramides A-C, a series of diketopiperazines (DKPs) with hydroxamic acid motifs. Feeding experiments showed that TamB catalyzes an unprecedented di-hydroxylation of the amide nitrogens in the diketopiperazine core. This tailoring reaction led to the formation of two bidentate iron-binding sites per molecule with an unusual iron-binding stoichiometry. The structure of the terramide A-Fe complex was characterized by liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy and electron paramagnetic resonance spectroscopy (EPR). Antimicrobial assays showed that the iron-binding motifs are crucial for the activity against bacteria and fungi. Murine infection experiments indicated that terramide production is crucial for the virulence of A. terreus and could be a potential antifungal drug target.

Design, Synthesis, and Antimicrobial Activity of Amide Derivatives Containing Cyclopropane.

As an important small organic molecule, cyclopropane is widely used in drug design. In this paper, fifty-three amide derivatives containing cyclopropane were designed and synthesized by introducing amide groups and aryl groups into cyclopropane through the active splicing method, and their antibacterial and antifungal activities were evaluated in vitro. Among them, thirty-five compounds were new compounds, and eighteen compounds were known compounds (F14, F15, F18, F20-F26, F36, and F38-F44). Bioassay results disclosed that four, three, and nine of the compounds showed moderate activity against Staphylococcus aureus, Escherichia coli, and Candida albicans, respectively. Three compounds were sensitive to Candida albicans, with excellent antifungal activity (MIC80 = 16 μg/mL). The molecular docking results show that compounds F8, F24, and F42 have good affinity with the potential antifungal drug target CYP51 protein.

S3.4e Unraveling the role of DOG genes in a novel alternative pathway of glycerol biosynthesis in Candida albicans and its influence on virulence

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMDOG genes, encoding for 2-deoxyglucose-6-phosphate phosphatase are low molecular weight phosphatases, with an unknown biological function. In contrast to Saccharomyces cerevisiae which has two DOG homologs, C. albicans only has one DOG gene. We hypothesized that DOG1 plays an important role under osmotic or ionic stress by biosynthesizing glycerol, which is known to be vital for biofilm formation and virulence of this pathogenic yeast, via a novel alternative pathway. The known classical pathway of glycerol production begins when the glycolytic intermediate molecule dihydroxyacetone phosphate (DHAP) is converted into glycerol-3-phosphate (G-3-P) by a pair of glycerol-3-phosphate-dehydrogenases, Gpd1 and Gpd2. G-3-P is further dephosphorylated into glycerol by glycerol-3-phosphate-phosphatases, Gpp1 and Gpp2. However, an alternative pathway, where DHAP is dephosphorylated into DHA, which is subsequently converted into glycerol has been proposed, but the enzymes involved in this process have not yet been described. We recently showed that in Saccharomyces cerevisiae, the DOG enzymes are involved in the production of DHA from DHAP, thereby allowing the synthesis of glycerol in the absence of the classical pathway. Overexpression of the DOG genes restored the osmo-tolerance of the gpp1Δ gpp2Δand gpd1Δ gpd2Δ double deletion strains. Furthermore, purified DOG enzymes could also convert DHAP into DHA (Awasthy et al., submitted). Since DOG1 has a potential role in biosynthesizing glycerol via an unconventional route, we are interested to determine its contribution in influencing virulence and biofilm formation in Candida albicans. This putative pathway has been overlooked for the past two decades, leaving behind an evident knowledge gap. We have now generated multiple deletion strains, using CRISPR Cas9, for the C. albicans counterparts of the GPP, GPD, and DOG genes as well as multiple DOG1 overexpression strains in which we observed the restoration of osmotic stress tolerance phenotypically and via growth curves. We also have NMR data showing the accumulation of various metabolites of central metabolism in these strains. Additionally, we have determined the possible role of DOG1 on virulence factors like morphogenesis, adhesion, and biofilm formation in vitro as well as in vivo, the latter with our catheter-based biofilm subcutaneous mouse model system. We also linked DOG1 and its role in glycerol biosynthesis to the survival of C. albicans inside macrophages. Finally, we would be setting up a high throughput small compound screening for this phosphatase as a potential antifungal drug target.

Phosphomannose Isomerase Is Involved in Development, Stress Responses, and Pathogenicity of Aspergillus flavus.

ABSTRACTAspergillus flavus causes invasive aspergillosis in immunocompromised patients and severe contamination of agriculturally important crops by producing aflatoxins. The fungal cell wall is absent in animals and is structurally different from that of plants, which makes it a potential antifungal drug target due to its essentiality for fungal survival. Mannose is one of the important components in the fungal cell wall, which requires GDP-mannose (GDP-Man) as the primary donor. Three consecutive enzymes, namely, phosphomannose isomerase (PMI), phosphomannose mutase (PMM), and GDP-mannose phosphorylase (GMPP), are required for GDP-Man biosynthesis. Thus, PMI is of prime importance in cell wall biosynthesis and also has an active role in sugar metabolism. Here, we investigated the functional role of PMI in A. flavus by generating a pmiA-deficient strain. The mutant required exogenous mannose to survive and exhibited reduced growth rate, impaired conidiation, early germination, disturbance in stress responses, and defects in colonization of crop seeds. Furthermore, attenuated virulence of the mutant was documented in both Caenorhabditis elegans and Galleria mellonella infection models. Our results suggested that PMI plays an important role in the development, stress responses, and pathogenicity of A. flavus and therefore could serve as a potential target for battling against infection and controlling aflatoxin contamination caused by A. flavus.IMPORTANCE Aspergillus flavus is a common fungal pathogen of humans, animals, and agriculturally important crops. It causes invasive aspergillosis in humans and also produces highly carcinogenic mycotoxins in postharvest crops that threaten food safety worldwide. To alleviate or eliminate the threats posed by A. flavus, it is necessary to identify genes involved in pathogenicity and mycotoxin contamination. However, little progress has been made in this regard. Here, we focused on PMI, which is the first enzyme involved in the biosynthesis pathway of GDP-Man and thus is important for cell wall synthesis and protein glycosylation. Our study revealed that PMI is important for growth of A. flavus. It is also involved in conidiation, germination, morphogenesis, stress responses, and pathogenicity of A. flavus. Thus, PMI is a potent antifungal target to curb the threats posed by A. flavus.

Molecular Characterization and the Essential Biological Function of the Metal Chaperone Protein MtmA in Aspergillus fumigatus.

The detoxification system of reactive oxygen species (ROS) plays critical roles in the survival and virulence of fungal pathogens in infected hosts, while superoxide dismutase (SOD) is the primary ROS scavenger. In the model yeast Saccharomyces cerevisiae, the metal chaperone protein Mtm1 is required for mitochondrial Sod2 activation and responses to oxidative stress. However, the function of the S. cerevisiae Mtm1 homolog in the human fungal pathogen Aspergillus fumigatus has not yet been clarified. In this study, we found that mitochondria-localized MtmA in A. fumigatus, a putative homolog of yeast Mtm1, not only has a similar function to Mtm1 in responding to oxidative stress resistance by affecting SodB (MnSOD) activity but is also essential for hyphal growth such that repressed expression of MtmA results in severe growth defects in A. fumigatus. In addition, the chelation of Zn2+ can obviously rescue growth defects caused by repression of MtmA, suggesting that MtmA may be involved in hyphal growth by affecting cellular Zn2+ detoxification. Moreover, MtmA contains four Mito-carr domains, whereas only the first Mito-carr domain is required for the function of MtmA. Therefore, the findings in this study suggest that MtmA in A. fumigatus has an important and unique function that is different from that in yeast. IMPORTANCE Knowledge of the key factors required for the viability of pathogenic fungi can help to explore new antifungal drugs. Here, we demonstrate that MtmA is involved in responding to oxidative stress by activating mitochondrial SodB activity. MtmA, especially for the first Mito-carr domain, is essential for colony growth by regulating cellular Zn2+ equilibrium and responses to oxidative stress in A. fumigatus. This is the first report of the vital and unique role of the MtmA protein in pathogenic fungi, indicating that it might be a potential antifungal drug target.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure.

The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.

Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential anti-fungal targets.

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2′-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2′-PO4 attacks NAD+ to form an RNA-2′-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential antifungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1Δ mutation and in vitro in NAD+-dependent conversion of a 2′-PO4 to a 2′-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD+, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2′-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2′-PO4 nucleotide with arabinose-2′-PO4 diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2′-phospho-ADP-ribosylated intermediate. Our results implicate the 2′-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.

Identification and characterization of a sulfite reductase gene and new insights regarding the sulfur-containing amino acid metabolism in the basidiomycetous yeast Cryptococcus neoformans.

The amino acid biosynthetic pathway of invasive pathogenic fungi has been studied as a potential antifungal drug target. Studies of the disruption of genes involved in amino acid biosynthesis have demonstrated the importance of this pathway in the virulence of Cryptococcus neoformans. Here, we identified the MET5 (CNL05500) and MET10 (CNG03990) genes in this pathway, both encoding sulfite reductase, which catalyzes the reduction of sulfite to sulfide. The MET14 (CNE03880) gene was also identified, which is responsible for the conversion of sulfate to sulfite. The use of cysteine as a sulfur source led to the production of methionine via hydrogen sulfide synthesis mediated by CYS4 (CNA06170), CYS3 (CNN01730), and MST1 (CND03690). MST1 exhibited high homology with the TUM1 gene of Saccharomyces cerevisiae, which has functional similarity with the 3-mercaptopyruvate sulfurtransferase (3-MST) gene in humans. Although the hypothesis that hydrogen sulfide is produced from cysteine via CYS4, CYS3, and MST1 warrants further study, the new insight into the metabolic pathway of sulfur-containing amino acids in C. neoformans provided here indicates the usefulness of this system in the development of screening tools for antifungal drug agents.

Emerging New Targets for the Treatment of Resistant Fungal Infections.

With the increasing morbidity and mortality of invasive fungal infections and the emergence of severe antifungal drug resistance, new drug targets and novel antifungal agents are urgently needed. Recently, better understanding of fungal pathogenesis has contributed to the rapid emergence of potential antifungal drug targets. This perspective aims to provide a comprehensive review of new antifungal targets and of medicinal chemistry efforts toward inhibitor discovery. Particular focus will be placed on the druggability of the targets and their potential to treat resistant fungal infections. Innovative strategies for the next generation of antifungal therapy, such as virulence factors, protein-protein interactions, and immune response-based proteins, will also be highlighted.

Antifungal drug testing by combining minimal inhibitory concentration testing with target identification by gas chromatography-mass spectrometry.

Fungal infections and their increasing resistance to antibiotics are an emerging threat to public health. Novel antifungal drugs, as well technologies that can help us bolster the antimicrobial pipeline and understand resistance mechanisms, are needed. The ergosterol biosynthetic pathway is one potential target for antifungal drugs. Here we describe how antifungal susceptibility testing can be combined with target identification in distal ergosterol biosynthesis by means of gas chromatography-mass spectrometry. The fungi are treated with sublethal doses of active components that block ergosterol biosynthesis, and the ergosterol biosynthesis intermediates are analyzed in a targeted metabolomics manner after derivatization (trimethylsilylation). Drug treatment results in distinct sterol patterns that are characteristic of the affected enzyme. Sterol identification based on relative retention times and electron ionization (EI) mass spectra, as well as semiquantitative assessment of ergosterol intermediates, is described. The protocol is applicable to yeasts and molds. The overall analysis time from incubation to test result is not more than 3 d. The assay can be used to determine whether an antifungal compound of interest targets sterol biosynthesis, and, if so, to determine which enzyme in the pathway it targets.

Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target.

Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.

Genomic Identification of Potential Targets Unique to Candida albicans for the Discovery of Antifungal Agents

Despite of modern antifungal therapy, the mortality rates of invasive infection with human fungal pathogen Candida albicans are up to 40%. Studies suggest that drug resistance in the three most common species of human fungal pathogens viz., C. albicans, Aspergillus fumigatus (causing mortality rate up to 90%) and Cryptococcus neoformans (causing mortality rate up to 70%) is due to mutations in the target enzymes or high expression of drug transporter genes. Drug resistance in human fungal pathogens has led to an imperative need for the identification of new targets unique to fungal pathogens. In the present study, we have used a comparative genomics approach to find out potential target proteins unique to C. albicans, an opportunistic fungus responsible for severe infection in immune-compromised human. Interestingly, many target proteins of existing antifungal agents showed orthologs in human cells. To identify unique proteins, we have compared proteome of C. albicans [SC5314] i.e., 14,633 total proteins retrieved from the RefSeq database of NCBI, USA with proteome of human and non-pathogenic yeast Saccharomyces cerevisiae. Results showed that 4,568 proteins were identified unique to C. albicans as compared to those of human and later when these unique proteins were compared with S. cerevisiae proteome, finally 2,161 proteins were identified as unique proteins and after removing repeats total 1,618 unique proteins (42 functionally known, 1,566 hypothetical and 10 unknown) were selected as potential antifungal drug targets unique to C. albicans.

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae.

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.

Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies

Aspergillus fumigatus is the main cause of severe invasive aspergillosis. To combat this life-threatening infection, only limited numbers of antifungals are available. The fungal alpha-aminoadipate pathway, which is essential for lysine biosynthesis, has been suggested as a potential antifungal drug target. Here we reanalyzed the role of this pathway for establishment of invasive aspergillosis in murine models. We selected the first pathway-specific enzyme, homocitrate synthase (HcsA), for biochemical characterization and for study of its role in virulence. A. fumigatus HcsA was specific for the substrates acetyl-coenzyme A (acetyl-CoA) and alpha-ketoglutarate, and its activity was independent of any metal ions. In contrast to the case for other homocitrate synthases, enzymatic activity was hardly affected by lysine and gene expression increased under conditions of lysine supplementation. An hcsA deletion mutant was lysine auxotrophic and unable to germinate on unhydrolyzed proteins given as a sole nutrient source. However, the addition of partially purified A. fumigatus proteases restored growth, confirming the importance of free lysine to complement auxotrophy. In contrast to lysine-auxotrophic mutants from other fungal species, the mutant grew on blood and serum, indicating the existence of high-affinity lysine uptake systems. In agreement, although the virulence of the mutant was strongly attenuated in murine models of bronchopulmonary aspergillosis, virulence was partially restored by lysine supplementation via the drinking water. Additionally, in contrast to the case for attenuated pulmonary infections, the mutant retained full virulence when injected intravenously. Therefore, we concluded that inhibition of fungal lysine biosynthesis, at least for disseminating invasive aspergillosis, does not appear to provide a suitable target for new antifungals.

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in the α-aminoadipate (AAA) pathway of lysine biosynthesis, which occurs in many fungi and certain archaea, and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is comprised of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed α/β-topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain covers the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex, the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Steady state kinetic assays and in vivo yeast growth assays on wild-type enzyme and active site mutants allow us to elucidate its catalytic mechanism, including the residues implicated in catalysis. Together these results yield new insights into the mechanism and regulation of HCS, which provide a platform to identify small molecule inhibitors of HCS that may be optimized and used as anti-fungal agents.

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed alpha/beta topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

Role of Nitrogen and Carbon Transport, Regulation, and Metabolism Genes for Saccharomyces cerevisiae Survival In Vivo

Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate, pyruvate, and fatty acids. Using mutants unable to either transport or utilize these compounds, we demonstrated that no individual nitrogen source was essential, while glucose was the most significant primary carbon source for yeast survival in vivo. Hydrolysis of the storage carbohydrate glycogen made a slight contribution for in vivo survival compared with a substantial requirement for trehalose hydrolysis. The ability to sense and respond to low glucose concentrations was also important for survival. In contrast, there was little or no requirement in vivo in this assay for any of the nitrogen-sensing pathways, nitrogen catabolite repression, the ammonium- or amino acid-sensing pathways, or general control. By using auxotrophic mutants, we found that some nitrogenous compounds (polyamines, methionine, and lysine) can be acquired from the host, while others (threonine, aromatic amino acids, isoleucine, and valine) must be synthesized by the pathogen. Our studies provide insights into the yeast-host environment interaction and identify potential antifungal drug targets.

Identification and assignment of function to the genes of Aspergillus fumigatus expressed at 37 degrees C.

Aspergillus fumigatus, a fungal pathogen, causes a spectrum of allergic and invasive disorders. In order to rapidly identify genes of this fungus relevant for pathogenesis and as potential antifungal drug targets, 125 expressed sequence tags (ESTs) were generated from 200 phage clones of a non-normalized cDNA library. Out of a novel 68 ESTs, 45 were assigned putative functions based on the sequence similarity. The identities of some of these genes suggest that they may be involved in pathogenesis or autoimmune reactions. Additional genes were identified that are possible targets for the development of antifungal drugs or that may be of use in diagnosing fungal infections.

Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis.

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and alpha-aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5 x 10(3) conidia per mouse. In this murine model, the Delta lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.