Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

In vivo analysis of septin heteropolymer rods and higher-order structures in filamentous fungi.

Individual septins from separate phylogenetic groups assemble into heteropolymer rods with defined compositions. Heteropolymer rods can in turn assemble into larger higher-order structures (HOS). Using a combination of immunoprecipitation and fluorescence microscopy in Aspergillus nidulans wild-type and septin-mutant strains, we have shown that even when septin HOS are not visible by fluorescence microscopy, heteropolymer rods can still be present and that a septin found largely in filamentous fungi is required for a subset of HOS. We raise several factors that are important for analysis of heteropolymer rods and HOS.

Growth control and polarization.

Filamentous fungi and yeasts both undergo polar growth. In Saccharomyces cerevisiae, where the mechanisms for polar growth are well-understood, polarity requires three steps: establishment of cortical markers specifying the site of bud emergence; relaying the bud site information via the Cdc42 Rho GTPase module; and recruitment of the morphogenetic machinery needed to remodel the cell surface to the specified site. Comparison of the genomes of Aspergillus fumigatus, A. nidulans and A. oryzae with that of S. cerevisiae show that the cortical markers are absent or poorly conserved, while the RhoGTPase signaling module and the morphogenetic machinery are highly conserved in the aspergilli. Genetic approaches to polarity using A. nidulans polarity mutants with defects in germ tube emergence (swo mutants) or branching (ahb mutants) will also be discussed.

Characterization of the Aspergillus nidulans septin (asp) gene family.

Members of the septin gene family are involved in cytokinesis and the organization of new growth in organisms as diverse as yeast, fruit fly, worm, mouse, and human. Five septin genes have been cloned and sequenced from the model filamentous fungus A. nidulans. As expected, the A. nidulans septins contain the highly conserved GTP binding and coiled-coil domains seen in other septins. On the basis of hybridization of clones to a chromosome-specific library and correlation with an A. nidulans physical map, the septins are not clustered but are scattered throughout the genome. In phylogenetic analysis most fungal septins could be grouped with one of the prototypical S. cerevisiae septins, Cdc3, Cdc10, Cdc11, and Cdc12. Intron-exon structure was conserved within septin classes. The results of this study suggest that most fungal septins belong to one of four orthologous classes.

Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus.

Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

Aspergillus nidulans swo mutants show defects in polarity establishment, polarity maintenance and hyphal morphogenesis.

When the spores of filamentous fungi break dormancy, they grow isotropically, adding cell wall material uniformly in every direction. Later they switch to polarized growth, with new material added to the tip of an emerging germ tube. To identify genes involved in the synthesis and localization of cell wall material in filamentous fungi, we screened a collection of temperature-sensitive Aspergillus nidulans mutants for swollen cells. We have isolated mutants representing eight genes involved in polarity establishment, polarity maintenance, and hyphal morphogenesis. On the basis of the results of temperature-shift experiments, swo C, D, and F are required to establish polarity, while swoA is required to maintain polarity. swo B, E, G, and H are involved in later hyphal morphogenesis. Our results suggest that polarity establishment and polarity maintenance are genetically separate events and that a persistent signal is required for apical extension in A. nidulans.

Relationship of actin, microtubules, and crosswall synthesis during septation in Aspergillus nidulans.

Studies of cytokinesis in animal cells demonstrate that microtubules play an important role in signaling the position of the actin-containing contractile ring and subsequent formation of the cleavage furrow. Septation in several fungi closely resembles animal cell cytokinesis in that a circumferential ring of actin is visible at the incipient division site. However, this does not necessarily mean that division is contractile since actin may also serve to localize septal wall synthesis. In addition, several studies in fission yeast have suggested that microtubules are dispensable for actin ring formation. We have used synchronized cells and fluorescence microscopy to follow actin structures, nuclear division and septal wall synthesis during septation in Aspergillus nidulans. Our data suggest that actin first appears at the septum site as a circumferential ring and that it later broadens and invaginates, forming an hourglass-shaped structure coincident with septal cell wall synthesis. Depolymerization of microtubules early in septation prevents circumferential actin ring formation. Depolymerization of microtubules after circumferential actin ring formation blocks both the progression to invaginating bands and septal wall synthesis. In contrast to studies in yeast cells, our data suggest that microtubules are required for both the initiation and progression of septation in A. nidulans.

The Aspergillus nidulans septin encoding gene, aspB, is essential for growth.

Septins are a highly conserved family of presumed cytoskeletal proteins involved in cytokinesis and morphogenesis in Saccharomyces cerevisiae and Drosophila melanogaster. To investigate the role of septins in filamentous fungi, we have identified presumptive septin homologues in Aspergillus nidulans. One of these septins, aspB, is expressed during vegetative growth and asexual sporulation. Based on cDNA and genomic sequences, the predicted aspB protein shares a P-loop motif and coiled coil regions with septins from other organisms. Antibodies generated against an aspB fusion protein recognized an A. nidulans protein of approximately 50 kDa, but could not localize the septin product in cells by immunofluorescence. Hybridization to a chromosome-specific ordered cosmid library placed the aspB gene on the right arm of chromosome I. Disruption of aspB showed that it is an essential gene.

Identification of three chitin synthase genes in the dimorphic fungal pathogen Sporothrix schenckii.

Degenerate PCR primers were used to amplify a 600-bp conserved gene region for chitin synthases from genomic DNA of Sporothrix schenckii, a dimorphic fungal pathogen of humans and animals. Three chitin synthase gene homologs were amplified as shown by DNA sequence analysis and by Southern blotting experiments. Based on differences among the predicted amino acid sequences of these homologs, each was placed within one of three different chitin synthase classes. Phylogenies constructed with the sequences and the PAUP 3.1.1 program showed that S. schenckii consistently clustered most closely with Neurospora crassa in each of the three chitin synthase classes. These findings are significant because the phylogenies support by a new method the grouping of the imperfect fungus S. schenckii with the Pyrenomycetes of the Ascomycota.

Chitin synthase-encoding gene(s) of the Zygomycete fungus Phycomyces blakesleeanus.

Using polymerase chain reaction (PCR) primers to two highly conserved sequences within fungal chitin synthase (CHS)-encoding genes, an approximately 750-bp DNA fragment was amplified from genomic DNA of Phycomyces blakesleeanus. The amino acid sequence deduced from the nucleotide sequence of this fragment best matches the motifs found in class-II CHS. The fragment includes an approximately 160-bp region that likely is an intron. Southern hybridization of restriction enzyme-digested genomic DNA, using the PCR-amplified DNA as the probe, suggests that P. blakesleeanus contains additional CHS-encoding genes (CHS). To our knowledge, this is the first report on the detection of a CHS gene in a Zygomycete fungus. These studies represent a major step toward exploring the molecular mechanisms of CHS regulation in Phycomyces. The prospects are exciting, since CHS is implicated to play a central role in the sensory responses of P. blakesleeanus involving growth modulations.

Classification of fungal chitin synthases.

Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHS1, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings.