[Show abstract][Hide abstract] ABSTRACT: Fungal meningitis is a serious disease caused by a fungal infection of the central nervous system (CNS) mostly in individuals with immune system deficiencies. Fungal meningitis is often fatal without proper treatment, and the mortality rate remains unacceptably high even with antifungal drug interventions. Currently, cryptococcal meningitis is the most common fungal meningitis in HIV-1/AIDS, and its disease mechanism has been extensively studied. The key steps for fungi to infect brain and cause meningitis after establishment of local infection are the dissemination of fungal cells to the bloodstream and invasion through the blood brain barrier to reach the CNS. In this review, we use cryptococcal CNS infection as an example to describe the current molecular understanding of fungal meningitis, including the establishment of the infection, dissemination, and brain invasion. Host and microbial factors that contribute to these infection steps are also discussed.
[Show abstract][Hide abstract] ABSTRACT: We recently observed that the micafungin MICs for some Candida glabrata fks hot spot mutant isolates are less elevated than those for the other echinocandins, suggesting that the efficacy of micafungin may be differentially dependent on such mutations. Three clinical C. glabrata isolates with or without (S3) fks hot spot mutations R83 (Fks2p-S663F) and RR24 (Fks1p-S629P) and low, medium, and high echinocandin MICs, respectively, were evaluated to assess the in vivo efficacy in an immunocompetent mouse model using three doses of each echinocandin. Drug concentrations were determined in plasma and kidneys by high-performance liquid chromatography (HPLC). A pharmacokinetic-pharmacodynamic mathematical model was used to define the area under the concentration-time curve (AUC) that produced half- and near-maximal activity. Micafungin was equally efficacious against the S3 and R83 isolates. The estimates for the AUCs of each echinocandin that induced half-maximal effect (E(50)s) were 194.2 and 53.99 mg · h/liter, respectively. In contrast, the maximum effect (E(max)) for caspofungin was higher against S3 than R83, but the estimates for E(50) were similar (187.1 and 203.5 mg · h/liter, respectively). Anidulafungin failed to induce a ≥1-log reduction for any of the isolates (AUC range, 139 to 557 mg · h/liter). None of the echinocandins were efficacious in mice challenged with the RR24 isolate despite lower virulence (reduced maximal growth, prolonged lag phase, and lower kidney burden). The AUC associated with half-maximal effect was higher than the average human exposure for all drug-dose-bug combinations except micafungin and the R83 isolate. In conclusion, differences in micafungin MICs are associated with differential antifungal activities in the animal model. This study may have implications for clinical practice and echinocandin breakpoint determination, and further studies are warranted.
Antimicrobial Agents and Chemotherapy 02/2012; 56(5):2435-42. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Candida glabrata is a leading cause of disseminated candidiasis. The echinocandins are increasingly used as first-line agents for the treatment of patients with this syndrome, although the optimal regimen for the treatment of invasive Candida glabrata infections in neutropenic patients is not known. We studied the pharmacokinetics (PK) and pharmacodynamics (PD) of micafungin, anidulafungin, and caspofungin in a neutropenic murine model of disseminated Candida glabrata infection to gain further insight into optimal therapeutic options for patients with this syndrome. A mathematical model was fitted to the data and used to bridge the experimental results to humans. The intravenous inoculation of Candida glabrata in mice was followed by logarithmic growth throughout the experimental period (101 h). A dose-dependent decline in fungal burden was observed following the administration of 0.1 to 20 mg/kg of body weight every 24 h for all three agents. The exposure-response relationships for each drug partitioned into distinct fungistatic and fungicidal components of activity. Surprisingly, the average human drug exposures following currently licensed regimens were predicted to result in a fungistatic antifungal effect. Higher human dosages of all three echinocandins are required to induce fungicidal effects in neutropenic hosts.
Antimicrobial Agents and Chemotherapy 08/2011; 55(10):4880-7. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The clinical utility of the echinocandins is potentially compromised by the emergence of drug resistance. We investigated whether Candida albicans with amino acid substitutions at position Ser645 in Fks1 can be treated with either a conventional or an elevated dosage of micafungin. We studied Candida albicans (wild-type SC5314; MIC, 0.06 mg/liter) and four fks1 mutants (one FKS1/fks1 heterozygote mutant [MIC, 0.5 mg/liter] and three fks1/fks1 homozygous mutants [MICs for all, 2 mg/liter]) with a variety of amino acid substitutions at Ser645. The pharmacokinetic and pharmacodynamic relationships were characterized in a persistently neutropenic murine model of disseminated candidiasis. A mathematical model was fitted to all pharmacokinetic and pharmacodynamic data. This mathematical model was then used to "humanize" the murine pharmacokinetics, and the predicted antifungal effect was determined. The estimated maximal rate of growth and ultimate fungal densities in the kidney for each of the strains were similar. The administration of micafungin at 1 mg/kg of body weight to the wild type resulted in moderate antifungal activity, whereas the administration of 5 and 20 mg/kg resulted in rapid fungicidal activity. In contrast, the FKS1/fks heterozygote was killed only with 20 mg/kg, and the homozygous fks1 mutants failed to respond to any dosage. The bridging study revealed that human dosages of 100 and 400 mg/day were active only against the wild type, with no activity against either the heterozygote or the homozygote mutants. Ser645 Fks1 Candida albicans mutants cannot be treated with either conventional or elevated dosages of micafungin and should be deemed resistant.
Antimicrobial Agents and Chemotherapy 07/2011; 55(7):3075-83. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptococcus neoformans is an AIDS-associated human fungal pathogen and the most common cause of fungal meningitis, with a mortality rate over 40% in AIDS patients. Significant advances have been achieved in understanding its disease mechanisms. Yet the underlying mechanism of a high frequency of cryptococcal meningitis remains unclear. The existence of high inositol concentrations in brain and our earlier discovery of a large inositol transporter (ITR) gene family in C. neoformans led us to investigate the potential role of inositol in Cryptococcus-host interactions. In this study, we focus on functional analyses of two major ITR genes to understand their role in virulence of C. neoformans. Our results show that ITR1A and ITR3C are the only two ITR genes among 10 candidates that can complement the growth defect of a Saccharomyces cerevisiae strain lacking inositol transporters. Both S. cerevisiae strains heterologously expressing ITR1A or ITR3C showed high inositol uptake activity, an indication that they are major inositol transporters. Significantly, itr1a itr3c double mutants showed significant virulence attenuation in murine infection models. Mutating both ITR1A and ITR3C in an ino1 mutant background activates the expression of several remaining ITR candidates and does not show more severe virulence attenuation, suggesting that both inositol uptake and biosynthetic pathways are important for inositol acquisition. Overall, our study provides evidence that host inositol and fungal inositol transporters are important for Cryptococcus pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: The CLSI established clinical breakpoints (CBPs) for caspofungin (CSF), micafungin (MCF) and anidulafungin (ANF) versus Candida. The same CBP (susceptible (S): MIC ≤ 2 mcg/ml; non-S: MIC > 2 mcg/ml) was applied to all echinocandins and species. More data now allow reassessment of these CBPs. We examined cases of echinocandin failure where both MICs and fks mutations were assessed; wild type (WT) MICs and epidemiological cutoff values (ECVs) for a large Candida collection; molecular analysis of fks hotspots for Candida with known MICs; and pharmacokinetic and pharmacodynamic (PK/PD) data. We applied these findings to propose new species-specific CBPs for echinocandins and Candida. Of 18 candidiasis cases refractory to echinocandins and with fks mutations, 28% (CSF), 58% (ANF) and 66% (MCF) had MICs in the S category using CBP of ≤ 2 mcg/ml, while 0-8% would be S using CBP of ≤ 0.25 mcg/ml. WT MIC distributions revealed ECV ranges of 0.03-0.25 mcg/ml for all major species except C. parapsilosis (1-4 mcg/ml) and C. guilliermondii (4-16 mcg/ml). Among Candida tested for fks mutations, only 15.7-45.1% of 51 mutants were detected using the CBP for NS of >2 mcg/ml. In contrast, a cutoff of >0.25 mcg/ml for C. albicans, C. tropicalis, C. krusei, and C. dubliniensis detected 85.6% (MCF) to 95.2% (CSF) of 21 mutant strains. Likewise, a cutoff of >0.12 mcg/ml for ANF and CSF and of >0.06 mcg/ml for MCF detected 93% (ANF) to 97% (CSF, MCF) of 30 mutant strains of C. glabrata. These data, combined with PK/PD considerations, support CBPs of ≤ 0.25 mcg/ml (S), 0.5 mcg/ml (I), ≥ 1 (R) for CSF/MCF/ANF and C. albicans, C. tropicalis and C. krusei and ≤ 2 mcg/ml (S), 4 mcg/ml (I), and ≥ 8 mcg/ml (R) for these agents and C. parapsilosis. The CBPs for ANF and CSF and C. glabrata are ≤ 0.12 mcg/ml (S), 0.25 mcg/ml (I), and ≥ 0.5 mcg/ml (R), whereas those for MCF are ≤ 0.06 mcg/ml (S), 0.12 mcg/ml (I), and ≥ 0.25 mcg/ml (R). New, species-specific CBPs for Candida and the echinocandins are more sensitive to detect emerging resistance associated with fks mutations, and better able to predict risk for clinical failure.
Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 02/2011; 14(3):164-76. · 12.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We now have a decade of experience with echinocandin drugs. Large-scale epidemiologic antifungal surveillance studies have
demonstrated that caspofungin, micafungin, and anidulafungin retain high potency on clinical isolates of Candida, and resistance remains relatively low. Yet reports of breakthrough infections involving strains with a high minimum inhibitory
concentration (MIC) are mounting. Mechanism-specific resistance involving amino acid substitutions in the Fks subunit(s) of
the drug target glucan synthase results in reduced enzyme sensitivity to drug and high MICs. The mechanism affects all three
drugs and is encountered in all Candida species, as well as in Aspergillus. An initial susceptibility testing breakpoint failed to adequately distinguish wild-type susceptible isolates from fks mutant resistant strains. Considering data from epidemiologic, microbiologic, pharmacokinetic/pharmacodynamic, biochemical,
and genetic studies that better capture resistant isolates with fks genotypes has resulted in a proposed new breakpoint which provides a more reliable measure of probable therapeutic success.
–Clinical breakpoint–Epidemiologic cutoff value
Current Fungal Infection Reports 01/2011; 5(3):113-119.
[Show abstract][Hide abstract] ABSTRACT: A high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument was developed to characterize the codon for glycine 54 in the cyp51A genes from 13 reference isolates and 12 clinical isolates of Aspergillus fumigatus. Mutations in this codon confer reduced susceptibility to itraconazole and posaconazole. The assay is simple to perform, and a result of "wild type" or "mutant" is available after approximately 1 h following DNA extraction using commercially available reagents and conventional primers.
Antimicrobial Agents and Chemotherapy 03/2010; 54(5):2248-51. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Opportunistic fungal infections are a major cause of morbidity and mortality in immunosuppressed patients; and in HIV-positive
individuals, infections due to Candida, Cryptococcus, and Pneumocystis are AIDS-defining illnesses. The widespread use of antifungal drugs, particularly triazole drugs, has led to the emergence
of primary resistance, which largely reflects infection with inherently less susceptible strains. Secondary resistance in
normally susceptible strains also occurs and involves a variety of mechanisms including target site modification and drug
efflux transporters. Resistance is a clinical management issue, but it has remained relatively constant in most developed
countries. In developing countries, resistance is minimal due to limited antifungal therapy. However, as access to these drugs
increases, it is particularly important to evaluate trends that reflect evolving resistance issues observed elsewhere, especially
among individuals with HIV/AIDS.
[Show abstract][Hide abstract] ABSTRACT: Antifungal drug resistance is a confounding factor that negatively impacts clinical outcome for patients with serious mycoses. Early detection of fungi in blood or other specimens with a rapid assessment of drug susceptibility could improve the survival of patients with invasive disease by accelerating the initiation of appropriate antifungal treatment. Recent years have seen the growth of molecular technology that is ideally suited for fungal identification and assessment of drug resistance mechanisms.
Elucidation of the genetic mechanisms responsible for triazole and echinocandin resistance in prominent Candida spp. and Aspergillus spp. provides an opportunity to develop molecular diagnostic platforms suitable for rapid detection of primary and secondary drug resistance. Several highly dynamic and robust amplification/detection methodologies are now available that can provide simultaneous species identification and high fidelity discrimination of resistance alleles.
Molecular diagnostic platforms are ideal for rapid detection of fungal pathogens and they provide an opportunity to develop in parallel molecular assays that can evaluate antifungal drug resistance.
Current Opinion in Infectious Diseases 10/2009; 22(6):568-73. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 microg/ml; voriconazole MIC, 2 microg/ml; ravuconazole MIC, 4 microg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 microg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the beta-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.
Antimicrobial Agents and Chemotherapy 10/2008; 52(10):3504-11. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Echinocandins target fungal beta-1,3 glucan synthesis and are used clinically to treat invasive aspergillosis. Although echinocandins do not completely inhibit in vitro growth of Aspergillus fumigatus, they do induce morphological changes in fungal hyphae. Because beta-1,3 glucans activate host antifungal pathways via the Dectin-1 receptor, we investigated the effect of echinocandins on inflammatory responses to A. fumigatus. Caspofungin- or micafungin-treated conidia and germlings induced less secretion of tumor necrosis factor (TNF) and CXCL2 by macrophages than did their untreated counterparts. Diminished secretion of TNF and CXCL2 correlated with diminished beta-glucan exposure on echinocandin-treated germ tubes. In contrast to treated conidia and germlings, echinocandin-treated hyphae stimulated increased release of TNF and CXCL2 by macrophages and demonstrated intense staining with a beta-glucan-specific antibody, particularly at hyphal tips. Our experiments demonstrate that echinocandin-induced morphological changes in A. fumigatus hyphae are accompanied by increased beta-glucan exposure, with consequent increases in Dectin-1-mediated inflammatory responses by macrophages.
The Journal of Infectious Diseases 08/2008; 198(2):176-85. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Invasive aspergillosis remains difficult to diagnose despite advances in imaging and antigen-based serological testing. To overcome this problem, nucleic acid (NA)-based amplification assays were introduced to identify infecting pathogens. Unfortunately, the reliability of such assays to detect Aspergillus spp. has met with mixed success. A new generation of NA platforms are emerging, which greatly improve our ability to detect Aspergillus-specific DNA and RNA from respiratory and blood samples. These platforms can accurately detect a single genome, and the emergence of pan-fungal and pan-Aspergillus probes offer promise for broader detection. PCR remains the most important platform, especially when coupled with real-time probes. It is multiplex friendly and can distinguish between closely related target sequences. Nucleic acid sequence-based amplification (NASBA) is an RNA-directed isothermal transcription-based amplification platform, which is more robust than PCR resulting in a 10(14)-fold amplification. RNA-based detection facilitates more target options and can be used to assess cell viability. Both DNA and RNA amplification platforms take advantage of allele-specific properties of probes, which are valuable for assessing drug resistance markers. Finally, as new molecular diagnostic platforms mature, their role may expand to include early monitoring of therapy.
Medical mycology: official publication of the International Society for Human and Animal Mycology 07/2008; 47 Suppl 1:S223-32. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial peptides (AMPs) are naturally occurring, broad-spectrum antimicrobial agents that have recently been examined for their utility as therapeutic antibiotics. Unfortunately, they are expensive to produce and are often sensitive to protease digestion. To address this problem, we have examined the activity of a peptide mimetic whose design was based on the structure of magainin, exhibiting its amphiphilic structure. We demonstrate that this compound, meta-phenylene ethynylene (mPE), exhibits antimicrobial activity at nanomolar concentrations against a variety of bacterial and Candida species found in oral infections. Since Streptococcus mutans, an etiological agent of dental caries, colonizes the tooth surface and forms a biofilm, we quantified the activity of this compound against S. mutans growing under conditions that favor biofilm formation. Our results indicate that mPE can prevent the formation of a biofilm at nanomolar concentrations. Incubation with 5 nM mPE prevents further growth of the biofilm, and 100 nM mPE reduces viable bacteria in the biofilm by 3 logs. Structure-function analyses suggest that mPE inhibits the bioactivity of lipopolysaccharide and binds DNA at equimolar ratios, suggesting that it may act both as a membrane-active molecule, similar to magainin, and as an intracellular antibiotic, similar to other AMPs. We conclude that mPE and similar molecules display great potential for development as therapeutic antimicrobials.
Antimicrobial Agents and Chemotherapy 12/2007; 51(11):4125-32. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: La résistance aux azolés est inhabituelle chez Aspergillus sp. Nous décrivons le cas d´un patient ayant reçu un traitement à long terme par itraconazole et voriconazole pour une aspergillose intra-cavitaire bilatérale chronique, avec des aspergillomes dont les isolats d´Aspergillus fumigatus ont développé une résistance simultanée à l´itraconazole et au voriconazole. Une nouvelle mutation (G138C) du géne cible (cyp51A) codant pour la 14-déméthylase a été détectée. Le patient a présenté une réponse à l´administration intraveineuse de casponfungine, qu´il a reçu six fois par semaine, sans apparition de résistance en 9 mois.
Journal De Mycologie Medicale - J MYCOLOGIE MEDICALE. 01/2007; 17.
[Show abstract][Hide abstract] ABSTRACT: Replacement of phenylalanine with leucine at position 391 in squalene epoxidase was identified as being responsible for terbinafine resistance in mutants of Aspergillus nidulans. The equivalent mutation was engineered into the ergA gene of Aspergillus fumigatus, resulting in an F389L substitution that also conferred resistance to this pathogenic mold.
Antimicrobial Agents and Chemotherapy 08/2006; 50(7):2533-6. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cell wall of human fungal pathogen Aspergillus fumigatus protects the fungus against threats from environment and interacts with the host immune system. Alpha(1-3)glucan is the major polysaccharide of Aspergillus fumigatus cell wall, and it has been shown to contribute to the virulence of diverse fungal pathogens. In A. fumigatus, three putative alpha(1-3)glucan synthase genes AGS1, AGS2 and AGS3 have been identified. AGS1 is responsible for cell wall alpha(1-3)glucan biosynthesis, but strains with deletions of either AGS1 or AGS2 are not defective in virulence [Beauvais, A., Maubon, D., Park, S., Morelle, W., Tanguy, M., Huerre, M., Perlin, D.S., Latgé, J. P., 2005. Two alpha(1-3) glucan synthases with different functions in Aspergillus fumigatus. Appl. Environ. Microbiol. 71, 1531-1538]. In contrast, we present evidence that AGS3 is also responsible for cell wall alpha(1-3)glucan biosynthesis and can modulate the virulence of A. fumigatus. An AGS3 deletion strain was found to produce faster and more robust disease than the parental strain in an experimental mouse model of aspergillosis. The apparent hyper-virulence in the AGS3-deleted mutant was correlated with an increased melanin content of the conidial cell wall, a better resistance to reactive oxygen species and a quicker germination rate. These results suggest an indirect role for AGS3 in virulence through an adaptive mechanism.
Fungal Genetics and Biology 06/2006; 43(5):366-75. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An association between reduced susceptibility to echinocandins and changes in the 1,3-beta-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.
Antimicrobial Agents and Chemotherapy 09/2005; 49(8):3264-73. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caspofungin acetate (CAS) is a member of a new class of clinically-approved echinocandin drugs to treat invasive aspergillosis. CAS inhibits the activity of beta-1,3-D-glucan synthase (GS), thus damaging the fungal cell wall. Although no clinical resistance of Aspergillus to CAS has been reported as yet, the development of in vitro reduced susceptibility is presumed to be inevitable. By contrast, echinocandin resistance in laboratory strains of Candida albicans and Saccharomyces cerevisiae has been well documented. To study the potential for clinical resistance in Aspergillus, two classes of Aspergillus fumigatus mutant strains were isolated that exhibited reduced susceptibility to CAS. In the first class, a site-directed mutation within the target gene (AfFKS1, encoding the putative catalytic subunit of GS) was introduced and shown to confer low-level (16-fold) reduced susceptibility. A second class of spontaneous mutants were sensitive to low levels of drug but displayed nearly normal growth above 0.5 microg/ml, suggesting induction of an unknown resistance mechanism. At higher levels of drug (> or = 16 microg/ml), the mutants displayed partially restored sensitivity. Preliminary studies indicate that neither target site mutations, nor changes in target gene expression are present in these strains, as has been documented for several yeasts. Instead, preliminary results indicate that the molecular mechanism(s) underlying reduced susceptibility of CAS in the A. fumigatus strains is novel, possibly due to remodeling of the cell wall components.
Medical Mycology 05/2005; 43 Suppl 1:S299-305. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alpha(1-3) glucan is a main component of the Aspergillus fumigatus cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in A. fumigatus, AGS1 and AGS2, are highly homologous to the AGS genes of Schizosaccharomyces pombe, which encode putative alpha(1-3) glucan synthases. The predicted Ags proteins of A. fumigatus have an estimated molecular mass of 270 kDa. AGS1 and AGS2 were disrupted in A. fumigatus. Both Deltaags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Deltaags1 presented a reduction in the alpha(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of A. fumigatus mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Deltaags1 and Deltaags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall alpha(1-3) glucan does not play a significant role in A. fumigatus pathogenicity.
Applied and Environmental Microbiology 04/2005; 71(3):1531-8. · 3.68 Impact Factor