Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence.

The opportunistic fungus Aspergillus fumigatus is the primary invasive mold pathogen in humans, and is responsible for an estimated 200,000 yearly deaths worldwide. Most fatalities occur in immunocompromised patients who lack the cellular and humoral defenses necessary to halt the pathogen's advance, primarily in the lungs. One of the cellular responses used by macrophages to counteract fungal infection is the accumulation of high phagolysosomal Cu levels to destroy ingested pathogens. A. fumigatus responds by activating high expression levels of crpA, which encodes a Cu+ P-type ATPase that actively transports excess Cu from the cytoplasm to the extracellular environment. In this study, we used a bioinformatics approach to identify two fungal-unique regions in CrpA that we studied by deletion/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse model of invasive pulmonary aspergillosis. Deletion of CrpA fungal-unique amino acids 1-211 containing two N-terminal Cu-binding sites, moderately increased Cu-sensitivity but did not affect expression or localization to the endoplasmic reticulum (ER) and cell surface. Replacement of CrpA fungal-unique amino acids 542-556 consisting of an intracellular loop between the second and third transmembrane helices resulted in ER retention of the protein and strongly increased Cu-sensitivity. Deleting CrpA N-terminal amino acids 1-211 or replacing amino acids 542-556 also increased sensitivity to killing by mouse alveolar macrophages. Surprisingly, the two mutations did not affect virulence in a mouse model of infection, suggesting that even weak Cu-efflux activity by mutated CrpA preserves fungal virulence.

Point Mutation or Overexpression of Aspergillus fumigatus cyp51B, Encoding Lanosterol 14α-Sterol Demethylase, Leads to Triazole Resistance.

Aspergillus fumigatus is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis. However, resistance, due primarily to cyp51 mutation, is rapidly increasing. A. fumigatus contains two Cyp51 isoenzymes, Cyp51A and Cyp51B. Overexpression and mutation of Cyp51A is a major cause of triazole resistance in A. fumigatus. The role of Cyp51B in generating resistance is unclear. Here, we show that overexpression or mutation of cyp51B results in triazole resistance. We demonstrate that introduction of a G457S Cyp51B mutation identified in a resistant clinical isolate results in voriconazole resistance in a naive recipient strain. Our results indicate that mutations in cyp51B resulting in clinical resistance do exist and should be monitored.

Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence.

Aspergillus fumigatus is the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis, A. fumigatus spores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened the Neurospora crassa gene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of the A. fumigatus rhomboid homolog rbdA resulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation-accurately phenocopying deletion of srbA. In vivo, rbdA deletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdA mutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease in A. fumigatus that mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation.

Identification and characterization of a novel family of selective antifungal compounds (CANBEFs) that interfere with fungal protein synthesis.

Invasive mycotic infections have become more common during recent decades, posing an increasing threat to public health. However, despite the growing needs, treatments for invasive fungal infections remain unsatisfactory and are limited to a small number of antifungals. The aim of this study was to identify novel fungal cell wall inhibitors from a library of small chemical compounds using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans sensitive to cell wall-active agents. Eight "hit" compounds affecting cell wall integrity were identified from a screen of 35,000 small chemical compounds. Five shared a common basic molecular structure of 4-chloro-6-arylamino-7-nitro-benzofurazane (CANBEF). The most potent compound, CANBEF-24, was characterized further and was shown to inhibit the growth of pathogenic Aspergillus, Candida, Fusarium, and Rhizopus isolates at micromolar concentrations but not to affect the growth of mammalian cell lines. CANBEF-24 demonstrated strong synergy in combination with caspofungin, an antifungal that inhibits cell wall biosynthesis. Genetic and biochemical analyses with Aspergillus nidulans and Saccharomyces cerevisiae indicated that CANBEFs selectively inhibit fungal rRNA maturation and protein synthesis, suggesting that their effect on the cell wall is indirect. CANBEFs were nontoxic in insect (Galleria mellonella, Drosophila melanogaster) and mouse models of fungal infection. Preliminary evidence showing no therapeutic benefit in these models suggests that further cycles of optimization are needed for the development of this novel class of compounds for systemic use.

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Horizontal Gene Transfer of Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary mold pathogen in humans. It can cause a wide range of diseases in humans, with high mortality rates in immunocompromised patients. The first-line treatments for invasive A. fumigatus infections are the triazole antifungals that inhibit Cyp51 lanosterol demethylase activity, blocking ergosterol biosynthesis. However, triazole-resistant strains of A. fumigatus are increasingly encountered, leading to increased mortality. The most common triazole resistance mechanisms in A. fumigatus are alterations in the cyp51A gene or promoter. We tested the hypothesis that A. fumigatus can acquire triazole resistance by horizontal gene transfer (HGT) of resistance-conferring gene cyp51A. HGT has not been experimentally analyzed in filamentous fungi. Therefore, we developed an HGT assay containing donor A. fumigatus strains carrying resistance-conferring mutated cyp51A, either in its chromosomal locus or in a self-replicating plasmid, and recipient strains that were hygromycin resistant and triazole sensitive. Donor and recipient A. fumigatus strains were cocultured and transferred to selective conditions, and the recipient strain tested for transferred triazole resistance. We found that chromosomal transfer of triazole resistance required selection under both voriconazole and hygromycin, resulting in diploid formation. Notably, plasmid-mediated transfer was also activated by voriconazole or hypoxic stress alone, suggesting a possible route to HGT of antifungal resistance in A. fumigatus, both in the environment and during host infection. This study provides, for the first time, preliminary experimental evidence for HGT mediating antifungal resistance in a pathogenic fungus. IMPORTANCE It is well known that bacteria can transfer antibiotic resistance from one strain to another by horizontal gene transfer (HGT), leading to the current worldwide crisis of rapidly emerging antibiotic-resistant bacteria. However, in fungi, HGT events have only been indirectly documented by whole-genome sequencing. This study directly examined fungal HGT of antibiotic resistance in a laboratory setting. We show that HGT of antifungal triazole resistance occurs in the important human fungal pathogen Aspergillus fumigatus. Importantly, we show a plasmid-mediated transfer of triazole resistance occurs under conditions likely to prevail in the environment and in infected patients. This study provides an experimental foundation for future work identifying the drivers and mechanistic underpinnings of HGT in fungi.

Therapy of murine pulmonary aspergillosis with antibody-alliinase conjugates and alliin.

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.

Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant.

OBJECTIVES: Despite the need for novel drugs to combat fungal infections, antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. The aim of this study was to screen a library of small chemical compounds to identify cell wall inhibitors using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans. This mutant is specifically susceptible to cell wall damaging compounds under PKC-repressive growth conditions. METHODS: The inhibitory effect of a library of small chemical compounds was examined in vitro using the conditional A. nidulans PKC strain and a panel of pathogenic fungal isolates. Microscopy was used to assess alterations to fungal ultrastructure following treatment. RESULTS: Three 'hit' compounds affecting cell wall integrity were identified from a screen of 5000 small chemical compounds. The most potent compound, CW-11, was further characterized and shown to specifically affect cell wall integrity. In clinical isolates of Aspergillus fumigatus, CW-11 induces morphological changes characteristic of damage to the cell wall, including wall thickening and rupturing. Analysis of the susceptibility of A. fumigatus and A. nidulans cell wall and signalling pathway mutants to CW-11 suggests that its mode of action differs from that of the antifungals caspofungin and voriconazole. CONCLUSIONS: This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.

In vitro synergy of caspofungin with licensed and novel antifungal drugs against clinical isolates of Fusarium spp.

Combinations of caspofungin (CAS) with amphotericin-B (AMB), voriconazole (VRC), terbinafine (TRB) and tacrolimus (FK-506) were tested in vitro with 10 Fusarium isolates. MIC and minimal effective concentrations (MEC) were investigated in accord with the CLSI methodology. Drug interactions were assessed by the fractional inhibitory concentration index (FICI). Synergy occurred in 10/10, 9/10, 7/10 and 4/10 isolates with CAS/FK-506, CAS/TRB, CAS/AMB and CAS/VRC, respectively. Caspofungin MECs reached clinically attainable concentrations with FK-506 and TRB. Hyphal length and DiBAC staining demonstrated enhanced inhibition and killing with CAS/FK-506 and CAS/TRB. The combination of CAS/TRB and CAS/FK-506 is strongly synergistic in vitro against Fusarium spp. Our finding should be further studied in animal models of invasive infections caused by this fungus.

Riboflavin and pantothenic acid biosynthesis are crucial for iron homeostasis and virulence in the pathogenic mold Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus is the most prevalent airborne fungal pathogen, causing invasive fungal infections mainly in immunosuppressed individuals. Death rates from invasive aspergillosis remain high because of limited treatment options and increasing antifungal resistance. The aim of this study was to identify key fungal-specific genes participating in vitamin B biosynthesis in A. fumigatus. Because these genes are absent in humans they can serve as possible novel targets for antifungal drug development. METHODS: By sequence homology we identified, deleted and analysed four key A. fumigatus genes (riboB, panA, pyroA, thiB) involved respectively in the biosynthesis of riboflavin (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6) and thiamine (vitamin B1). RESULTS: Deletion of riboB, panA, pyroA or thiB resulted in respective vitamin auxotrophy. Lack of riboflavin and pantothenic acid biosynthesis perturbed many cellular processes including iron homeostasis. Virulence in murine pulmonary and systemic models of infection was severely attenuated following deletion of riboB and panA, strongly reduced after pyroA deletion and weakly attenuated after thiB deletion. CONCLUSIONS: This study reveals the biosynthetic pathways of the vitamins riboflavin and pantothenic acid as attractive targets for novel antifungal therapy. Moreover, the virulence studies with auxotrophic mutants serve to identify the availability of nutrients to pathogens in host niches. ABBREVIATIONS: BPS: bathophenanthrolinedisulfonate; BSA: bovine serum albumin; CFU: colony forming unit; -Fe: iron starvation; +Fe: iron sufficiency; hFe: high iron; NRPSs: nonribosomal peptide synthetases; PKSs: polyketide synthaseses; wt: wild type.

The Aspergillus nidulans pkcA gene is involved in polarized growth, morphogenesis and maintenance of cell wall integrity.

The protein kinase C (PKC) family participates in maintaining integrity and growth of fungal cell walls. However, the precise molecular role of these proteins in the filamentous fungi remains unknown. In this work, pkcA, the gene encoding the PKC homolog in the filamentous fungus Aspergillus nidulans, was cloned and its function analyzed using a conditional alcA-PKC mutant strain. Repression of pkcA expression resulted in increased conidial swelling, decreased rates of hyphal growth, changes in the ultrastructure of the cell wall and increased sensitivity to antifungal agents. These results suggest that the protein encoded by pkcA is involved in key aspects of cell morphogenesis and cell wall integrity.

Coding tandem repeats generate diversity in Aspergillus fumigatus genes.

Genes containing multiple coding mini- and microsatellite repeats are highly dynamic components of genomes. Frequent recombination events within these tandem repeats lead to changes in repeat numbers, which in turn alters the amino acid sequence of the corresponding protein. In bacteria and yeasts, the expansion of such coding repeats in cell wall proteins is associated with alterations in immunogenicity, adhesion, and pathogenesis. We hypothesized that identification of repeat-containing putative cell wall proteins in the human pathogen Aspergillus fumigatus may reveal novel pathogenesis-related elements. Here, we report that the genome of A. fumigatus contains as many as 292 genes with internal repeats. Fourteen of 30 selected genes showed size variation of their repeat-containing regions among 11 clinical A. fumigatus isolates. Four of these genes, Afu3g08990, Afu2g05150 (MP-2), Afu4g09600, and Afu6g14090, encode putative cell wall proteins containing a leader sequence and a glycosylphosphatidylinositol anchor motif. All four genes are expressed and produce variable-size mRNA encoding a discrete number of repeat amino acid units. Their expression was altered during development and in response to cell wall-disrupting agents. Deletion of one of these genes, Afu3g08990, resulted in a phenotype characterized by rapid conidial germination and reduced adherence to extracellular matrix suggestive of an alteration in cell wall characteristics. The Afu3g08990 protein was localized to the cell walls of dormant and germinating conidia. Our findings suggest that a subset of the A. fumigatus cell surface proteins may be hypervariable due to recombination events in their internal tandem repeats. This variation may provide the functional diversity in cell surface antigens which allows rapid adaptation to the environment and/or elusion of the host immune system.

Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence.

The ECM33/SPS2 family of glycosylphosphatidylinositol-anchored proteins plays an important role in maintaining fungal cell wall integrity and virulence. However, the precise molecular role of these proteins is unknown. In this work, AfuEcm33, the gene encoding the ECM33 homologue in the important pathogenic fungus Aspergillus fumigatus, has been cloned and its function analysed. It is shown that disruption of AfuEcm33 results in rapid conidial germination, increased cell-cell adhesion, resistance to the antifungal agent caspofungin and increased virulence in an immunocompromised mouse model for disseminated aspergillosis. These results suggest that the protein encoded by AfuEcm33 is involved in key aspects of cell wall morphogenesis and plays an important role in A. fumigatus virulence.

Analysis of the Aspergillus nidulans thaumatin-like cetA gene and evidence for transcriptional repression of pyr4 expression in the cetA-disrupted strain.

The asexual spore or conidium plays a critical role in the life cycle of many filamentous fungi. However, the process of conidial germination remains surprisingly obscure. To better understand this process at the molecular level we characterized the Aspergillus nidulans cetA gene which is uniquely transcribed in conidiating cultures and whose transcript is significantly enriched in mature conidia. CetA is a member of a novel family of fungal genes of unknown function with homology to plant thaumatin-like (PR-5) defense proteins. We demonstrate by Northern analysis that cetA is a glucose-repressible gene. Transcriptional repression is dependent on the presence of protein kinase A. Western analysis indicates that the CETA protein is absent from conidia but is highly expressed during the first 6h of germination and is secreted into the medium. Disruption of the cetA gene seemingly results in delayed germination, slow growth, abnormal hyphal branching, and cell-wall defects. However, further analysis shows that the mutant phenotype is the result of glucose-dependent transcriptional repression of the pyr4 selectable marker used to disrupt the cetA gene. This is the first time that repression of a selectable marker ("position effect") has been reported in A. nidulans, a finding that may well be of significance in the analysis and interpretation of mutant phenotypes in this organism.

Use of the Porcine Intestinal Epithelium (PIE)-Assay to analyze early stages of colonization by the human fungal pathogen Candida albicans.

Virulence of C. albicans strains can be tested using a mouse model of haematogenously disseminated Candida cells. Initial steps of host-pathogen contact such as adhesion and colonization are not taken into account due to the injection of Candida cells into the blood stream. Here we describe an assay, based on the ex vivo usage of porcine intestinal epithelium (PIE), that is useful to monitor the early stages of a C. albicans infection. The ability of C. albicans to undergo morphogenetic switching between yeast and hyphal stages is thought to contribute to its virulence. We found that hyphal formation was required to allow cells to colonize the PIE. The non-filamentous mutant strains efg1/cph1 which lacks two of the central transcription factors that are required to promote hyphal growth and wal1 that carries a deletion of the C. albicans homolog of the human Wiskott-Aldrich Syndrome Protein and is deficient in endocytosis showed only weak adherence. Furthermore, the wal1 mutant was found to be reduced in virulence using the mouse tail vein injection assay. We also analyzed the colonization properties of a variety of other mutant strains carrying deletions of either secreted aspartyl proteinase (SAP)-family genes or amino acid permease encoding genes (GAP1, SSY1, and PUT4). Interestingly, the nag5 strain which lacks an N-acetylglucosamine kinase showed enhanced filamentation and invasive growth as well as increased resistance against farnesol.

Disruption of the Aspergillus fumigatus argB gene using a novel in vitro transposon-based mutagenesis approach.

We disrupted the Aspergillus fumigatus argB gene, encoding ornithine transcarbamylase, using a novel in vitro transposon-based mutagenesis approach. This approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Using this approach, we obtained arginine auxotrophs of A. fumigatus. Full characterization of the argB insertion was performed by Southern blot analysis. These strains can be supplemented by addition of arginine into the culture medium and can be fully rescued to arginine prototrophy by transformation with the intact A. fumigatus argB gene.

In vitro synergy of caspofungin and itraconazole against Aspergillus spp.: MIC versus minimal effective concentration end points.

Caspofungin and itraconazole were studied alone and in combination against 31 clinical isolates of Aspergillus spp. according to NCCLS M38-P guidelines. MICs and microscopic minimal effective concentrations (MECs) were recorded, and synergy was calculated by using both end points. Synergy or synergy to additivity was found in 30 of 31 isolates by using MIC end points. With MEC end points no synergy was found and indifference was detected in 26 of 31 strains.

In vitro activity of caspofungin combined with sulfamethoxazole against clinical isolates of Aspergillus spp.

Caspofungin (CAS) inhibits fungal cell wall synthesis. Sulfamethoxazole (SMX) inhibits folate biosynthesis and is active in vitro against Aspergillus spp. We studied the activities of the combination of CAS and SMX against 31 Aspergillus isolates and compared them with that of SMX combined with amphotericin B (AMB) or itraconazole (ITC). MICs and minimal effective concentrations (MECs) were determined by the NCCLS broth microdilution method. With MIC endpoints, the combination of SMX and CAS showed synergy or synergy to additivity against 29 of 31 isolates. With MEC endpoints, synergy to additivity was found against 12 of 31 isolates and indifference was displayed against the rest of them. SMX in combination with AMB or ITC was not truly synergistic, while synergy to additivity was found for SMX-AMB and SMX-ITC against 17 of 31 and 3 of 12 isolates, respectively. No antagonism was found with any of the drug combinations. Further analysis of the synergy of CAS and SMX was performed by detailed measurement of hyphal length by microscopy and time-dependent 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based hyphal damage experiments. With MEC endpoints, the combination of CAS and SMX was characterized by a greater than 50% decrease in hyphal length compared to the hyphal lengths achieved with double the concentration of each drug alone. The XTT-based hyphal damage studies showed a statistically significant (P < 0.05) reduction in viability with CAS and SMX in combination compared to the viabilities achieved with double the concentration of each drug alone. These findings support the synergy results found by using MIC endpoints and suggest that visual MEC measurements may not be sufficient to identify the synergistic interactions seen by more sensitive, quantitative methods. Animal models are required to validate the significance of the synergy of CAS and SMX against Aspergillus spp. observed in vitro.

Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae.

The antifungal agent caspofungin (CAS) specifically interferes with glucan synthesis and cell wall formation. To further study the cellular processes affected by CAS, we analyzed a Saccharomyces cerevisiae mutant collection (4,787 individual knockout mutations) to identify new genes affecting susceptibility to the drug. This collection was screened for increased CAS sensitivity (CAS-IS) or increased CAS resistance (CAS-IR). MICs were determined by the broth microdilution method. Disruption of 20 genes led to CAS-IS (four- to eightfold reductions in the MIC). Eleven of the 20 genes are involved in cell wall and membrane function, notably in the protein kinase C (PKC) integrity pathway (MID2, FKS1, SMI1, and BCK1), chitin and mannan biosynthesis (CHS3, CHS4, CHS7, and MNN10), and ergosterol biosynthesis (ERG5 and ERG6). Four of the 20 genes (TPO1, VPS65, VPS25, and CHC1) are involved in vacuole and transport functions, 3 of the 20 genes (CCR4, POP2, and NPL3) are involved in the control of transcription, and 2 of the 20 genes are of unknown function. Disruption of nine additional genes led to CAS-IR (a fourfold increase of MIC). Five of these nine genes (SLG1, ERG3, VRP1, CSG2, and CKA2) are involved in cell wall function and signal transduction, and two of the nine genes (VPS67 and SAC2) are involved in vacuole function. To assess the specificity of susceptibility to CAS, the MICs of amphotericin B, fluconazole, flucytosine, and calcofluor for the strains were tested. Seven of 20 CAS-IS strains (with disruption of FKS1, SMI1, BCK1, CHS4, ERG5, TPO1, and ILM1) and 1 of 9 CAS-IR strains (with disruption of SLG1) demonstrated selective susceptibility to CAS. To further explore the importance of PKC in CAS susceptibility, the activity of the PKC inhibitor staurosporine in combination with CAS was tested against eight Aspergillus clinical isolates by the microdilution assay. Synergistic or synergistic-to-additive activities were found against all eight isolates by use of both MIC and minimum effective concentration endpoints.

Efficacy of allicin, the reactive molecule of garlic, in inhibiting Aspergillus spp. in vitro, and in a murine model of disseminated aspergillosis.

OBJECTIVES: The evaluation of allicin, the biologically active compound responsible for the antimicrobial activities of freshly crushed garlic cloves, in inhibiting Aspergillus spp. in vitro and in a murine model of disseminated aspergillosis. METHODS: Pure allicin was prepared by reacting synthetic alliin with a stabilized preparation of the garlic enzyme alliinase. We tested the in vitro efficacy of pure allicin against 31 clinical isolates of Aspergillus spp. using a microdilution broth method and following the NCCLS guidelines (document M-38P). Subsequently, the in vivo efficacy of allicin was tested in immunocompetent mice infected intravenously (iv) with Aspergillus fumigatus conidia. Allicin (5 mg/kg body weight) was administered iv once daily for 5 days post-infection or orally (po) (9 mg/kg body weight) for 5 days pre-infection and 10 days post-infection. No ill effects were observed in allicin-treated uninfected mice. RESULTS: The in vitro MICs and MFCs of allicin were between 8 and 32 mg/L, indicating that allicin in its pure form may be an effective fungicide in vitro. Time-kill studies indicate that allicin exerts its fungicidal activity within 2-12 h of administration in vitro. Allicin treatment significantly prolonged survival of infected mice (P < 0.01) from mean survival time (MST) = 7.7 days in untreated mice to MST = 21.3 and 13.9 days for allicin iv and po treated mice, respectively. Allicin iv treatment led to a significant (P < 0.001) 10-fold reduction in fungal burden in A. fumigatus infected mice as evaluated by quantitative fungal cultures of kidney tissue samples. CONCLUSIONS: These favourable results, despite the short half-life of this compound in vivo, support further studies of controlled sustained release or more prolonged administration of allicin as a treatment for aspergillosis.