The RAM network in pathogenic fungi

The regulation of Ace2 and morphogenesis (RAM) network is a protein kinase signaling pathway conserved among eukaryotes from yeasts to humans. Among fungi, the RAM network has been most extensively studied in the model yeast Saccharomyces cerevisiae and has been shown to regulate a range of cellular processes, including daughter cell-specific gene expression, cell cycle regulation, cell separation, mating, polarized growth, maintenance of cell wall integrity, and stress signaling. Increasing numbers of recent studies on the role of the RAM network in pathogenic fungal species have revealed that this network also plays an important role in the biology and pathogenesis of these organisms. In addition to providing a brief overview of the RAM network in S. cerevisiae, we summarize recent developments in the understanding of RAM network function in the human fungal pathogens Candida albicans, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Pneumocystis spp.     

Growth inhibition of Candida species and Aspergillus fumigatus by statins

Statins are a class of drugs widely used for lowering high cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of cholesterol. We studied the effects of two major statins, simvastatin and atorvastatin, on five Candida species and Aspergillus fumigatus. The statins strongly inhibited the growth of all species, except Candida krusei. Supplementation of Candida albicans and A. fumigatus with ergosterol or cholesterol in aerobic culture led to substantial recovery from the inhibition by statins, suggesting specificity of statins for the mevalonate synthesis pathway. Our findings suggest that the statins could have utility as antifungal agents and that fungal colonization could be affected in those on statin therapy.     

Antifungal activity of microbial secondary metabolites

Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi.     

Cryptic speciation in the cosmopolitan and clonal human pathogenic fungus Aspergillus fumigatus

Microbes and other organisms smaller than one to a few millimeters in size are hypothesized to have global populations, in contrast to the geographically restricted ranges of larger organisms. However, fungi, which routinely have reproductive propagules no larger than 10 micrometers, challenge the generality of this hypothesis because recent studies have shown that globally distributed morphological species embrace two or more geographically restricted phylogenetic species. We used the concordance of gene genealogies to recognize phylogenetic species in the globally distributed opportunistic human pathogenic fungus, Aspergillus fumigatus. Based on DNA sequence data of five loci for each of 63 individuals collected from five continents, we have delineated two phylogenetic species in this single morphological species. Unlike all other fungi examined to date, both genetically isolated groups showed a global distribution with no evidence of a correlation between genotype and geographic location. Sexual reproduction has never been observed in A. fumigatus, but when the same data were used to explore the association of alleles at the five loci for one of the phylogenetic species, evidence was found to support recombination. The discovery of a cryptic species is medically relevant because different species are likely to differ in virulence or drug resistance. The discovery of a globally distributed A. fumigatus species clade highlights the need for ecological studies of the fungus to either document global dispersal or propose alternative mechanisms by which it persists as single, global phylogenetic population.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Improvements of PCR-based identification targeting the DNA topoisomerase II gene to determine major species of the opportunistic fungi Candida and Aspergillus fumigatus

For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.     

Aspergillus fumigatus: saprophyte or pathogen?

Large-scale genome comparisons have shown that no gene sets are shared exclusively by both Aspergillus fumigatus and any other human pathogen sequenced to date, such as Candida or Cryptococcus species. By contrast, and in agreement with the environmental occurrence of this fungus in decaying vegetation, the enzymatic machinery required by a fungus to colonize plant substrates has been found in the A. fumigatus genome. In addition, the proteome of this fungus contains numerous efflux pumps, including >100 major facilitators that help the fungus to resist either natural aggressive molecules present in the environment or antifungal drugs in humans. Environment sensing, counteracting reactive oxidants, and retrieving essential nutriments from the environment are general metabolic traits that are associated with the growth of the saprotrophic mold A. fumigatus in an unfriendly environment such as its human host.     

FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.     

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.     

Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.     

The application of immunoperoxidase staining for the detection of causative fungi in tissue specimens of mycosis I

This study was performed in order to identify the fungi of four species (Aspergillus fumigatus, Fusarium anthophilum, Candida albicans, Cryptococcus neoformans) in formalin-fixed, paraffin-embedded tissue sections by the indirect method of immunoperoxidase staining. Mature albino rabbits were immunized by formalin-killed organisms. The antibodies were prepared by precipitation at a 50% saturation of ammonium sulfate and were checked for cross-reactivities by Ouchterlony's double immunodiffusion and precipitin test. The immunoperoxidase staining was applied to the paraffin-embedded tissue sections of infected mice, human autopsy and biopsy specimens. Although each fungus was stained clearly the cell wall, cross-reactivities appeared among them, however it was possible to identify four fungi by absorption and dilution of the antisera.     

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum

AIMS: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed. METHODS AND RESULTS: An extract from a Trichoderma species showed a more potent activity in an agar-based assay against the null mutant fks1::HIS strain than against the wild-type strain, suggesting that it could contain a glucan synthesis inhibitor. The active component was identified as the known compound ergokonin A. The compound exhibited activity against Candida and Aspergillus species, but was inactive against Cryptococcus species. It induced alterations in the hyphal morphology of Aspergillus fumigatus. The identification of the producing isolate was confirmed by sequencing of the rDNA internal transcribed spacers and comparison with the sequences of other Trichoderma species. The analysis showed that the producing fungus had a high homology with other strains classified as Trichoderma longibrachiatum and its teleomorph Hypocrea schweinitzii. CONCLUSIONS: The antifungal activity spectrum of ergokonin A and the morphology alterations induced on A. fumigatus are consistent with glucan synthesis as the target for ergokonin A. The production of ergokonin A is not uncommon, but is probably restricted to Trichoderma species. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery that ergokonin A could be an inhibitor of glucan synthesis, having a structure very different to other inhibitors, increases the likelihood that orally active agents with this fungal-specific mode of action may be developed.     

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.     

Protein phosphatase Z modulates oxidative stress response in fungi

The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.     

Essential gene identification and drug target prioritization in Aspergillus fumigatus

Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BDelta, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors.     

Structural and kinetic differences between human and Aspergillus fumigatus D-glucosamine-6-phosphate N-acetyltransferase

Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Appearance of Colonies of Prototheca on CHROMagar Candida medium

The microorganisms capable of producing opportunist infections include the yeast-like organisms of the genus Candida, and the unicellular algae of the genus Prototheca, which share common features and can, therefore, lead to confusion. Their colonies are almost identical and they grow in the same culture media used routinely in mycology. CHROMagar Candida is a new chromogenic differential isolation medium that facilitates the presumptive differentiation of some of the most clinically important yeast-like organisms. To our knowledge, the use of CHROMagar Candida with Prototheca spp. has not been reported in the literature. This report describes the growth of 151 strains of Prototheca on CHROMagar Candida compared to the growth of a total of 326 well-characterized yeast organisms of the genera Candida, Cryptococcus, Trichosporon, Geotrichum, and Saccharomyces. It is clinically relevant to note that algae of the genus Prototheca (P. wickerhamii, P. zopfii, and P. stagnora) and of the genus Candida parapsilosis produced similar cream-colored colonies on CHROMagar Candida medium. Based on their growth on CHROMagar, a new species of Candida is described, C. zeylanoides, which has blue-green colonies. The colonies of two species of Trichosporon are also differentiated: the blue-green colonies of T. beigelii and the pink colonies of T. capitatum. This revised version was published online in August 2006 with corrections to the Cover Date.