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Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival, and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains...
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... were echinulated with a dense and typical melanin outer layer in both parental and mutant strains. Moreover, the germination capacity of the conidia produced on Malt-agar media at room temperature was not affected and the germination kinetics of DmybA and parental conidia were similar on Sabouraud-agar medium (Supporting Information Fig. S7). ...
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... addition, the RNA-seq data indicated that the trehalose biosynthetic process (GO 0005992) was significantly overrepresented in the genes with a GFOLD lower than 21.5 (Table 2). The trehalose content of 9-day-old conidia was therefore quantified in the DmybA mutant and compared with that of the DvelC, DvelB, DveA, DvosA, DwetA and DcspA mutants ( Fig. 7A; Supporting Information Fig. S15). The DwetA and DcspA mutants had the lowest amount of trehalose, 13% and 40% of the parental strain respectively. To a lesser but to a significant extent DmybA, DvelB and DvosA had a significant reduction of the trehalose content (about 50%) compared with the parental strain. These data were in ...
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... other conidial polyols varied differently from the trehalose in the DmybA, DvelC, DvelB, DveA, DvosA, DwetA and DcspA mutants (Fig. 7B). The DwetA and DcspA mutants had a very high amount of mannitol (>33 the concentration of the parental strain) whereas the DvosA, DvelB and DmybA mutants showed only a slight difference in the mannitol level with respect to the parental strain. DcspA, DwetA and DmybA mutants had a very low amount of arabitol whereas the DvosA and DvelB ...
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... very high amount of mannitol (>33 the concentration of the parental strain) whereas the DvosA, DvelB and DmybA mutants showed only a slight difference in the mannitol level with respect to the parental strain. DcspA, DwetA and DmybA mutants had a very low amount of arabitol whereas the DvosA and DvelB had an amount similar to the parental strain (Fig. 7C). These results suggest that the conidial intracellular osmolytes are controlled by compensatory pathways, which are differently activated in the different conidiating mutant ...
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... DcspA mutant was constructed in the 1163/CEA17 DakuB KU80 to have the background same as the mybA deletion while the mutant previously analyzed by Levdansky et al. (2007) was constructed in a Afu293 background.The parental strain CEA17_DakuB ku80 was transformed by homologous recombination with a fused gDNA containing the cspA 5 0 upstream border, the hygromycin cassette and the cspA 3 0 downstream. Transformants were selected in 150 lg/ml of hygromycin and their gDNA was used to confirm the right and unique insertion in their genome by PCR and Southern blot techniques (Supporting Information Fig. S17). ...
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... conidiation was strongly reduced in the DmybA mutant, scanning and transmission electron microscopy studies showed that the DmybA mutant pro- duced conidia which looked morphologically similar to the parental strain (Supporting Information Fig. S6). Conidia were echinulated with a dense and typical mela- nin outer layer in both parental and mutant strains. Moreover, the germination capacity of the conidia pro- duced on Malt-agar media at room temperature was not affected and the germination kinetics of DmybA and parental conidia were similar on Sabouraud-agar medium (Supporting Information Fig. ...
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... of the DcspA strain. A DcspA mutant was constructed in the 1163/CEA17 DakuB KU80 to have the background same as the mybA deletion while the mutant previously analyzed by Levdansky et al. (2007) was con- structed in a Afu293 background.The parental strain CEA17_DakuB ku80 was transformed by homologous recombination with a fused gDNA containing the cspA 5 0 upstream border, the hygromycin cassette and the cspA 3 0 downstream. Transformants were selected in 150 lg/ml of hygromycin and their gDNA was used to confirm the right and unique insertion in their genome by PCR and Southern blot techniques (Supporting Information Fig. ...
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... conidial phenotype of the DmybA strain was remi- niscent of several mutants that all showed a reduction (or even complete loss) of the conidial trehalose content associated with a decrease in conidial viability ( Tao and Yu, 2011;Park et al., 2012aPark et al., , 2012b). In addition, the RNA-seq data indicated that the trehalose biosynthetic process (GO 0005992) was significantly overrepre- sented in the genes with a GFOLD lower than 21.5 (Table 2). The trehalose content of 9-day-old conidia was therefore quantified in the DmybA mutant and com- pared with that of the DvelC, DvelB, DveA, DvosA, DwetA and DcspA mutants ( Fig. 7A; Supporting Infor- mation Fig. S15). The DwetA and DcspA mutants had the lowest amount of trehalose, 13% and 40% of the parental strain respectively. To a lesser but to a signifi- cant extent DmybA, DvelB and DvosA had a significant reduction of the trehalose content (about 50%) com- pared with the parental strain. These data were in agreement with a tight regulation of these trehalose genes by the mybA gene. In contrast, the deletion of velC and veA genes which are not controlled by mybA, was not associated with a reduction in conidial trehalose in the respective mutant strains. The two methods used to quantify the amount of trehalose gave the same tre- halose amount (Fig. 7, Supporting Information Fig. S15). This result was in agreement with the RNA-seq data that showed that the cspA, wetA, velB and vosA genes were controlled (at least partially) by MybA pro- tein. Moreover, it indicated that MybA was involved in the regulation of the trehalose metabolism. In A. fumiga- tus, two different pathways have been proposed to be involved in the trehalose biosynthesis ( Lamarre et al., 2008;Al-Bader et al., 2010;Puttikamonkul et al., 2010). In the first pathway, glucose-6-phosphate and UDP- glucose are transformed by trehalose-6-phosphate syn- thases into trehalose-6-phosphate and a phosphate group is removed from the trehalose-6-phosphate by a trehalose-6-phosphate phosphatase to yield trehalose. In silico GFOLD analysis has shown that most genes assigned to the trehalose-6-P-synthases and phospha- tase complex (AFUA_6G12950, AFUA_2G04010, AFUA_2G04020, AFUA_4G03190 and AFUA_5G14300, AFUA_3G05650, AFUA_7G03940) were significantly down regulated (Table 2). This down regulation of the genes involved in the trehalose pathway which has been noticed in the RNA-seq data (Table 2) was confirmed by qRT-PCR experiments (data not shown). In the second pathway involved in the synthesis of trehalose in A. fumigatus, trehalose phosphorylases produce trehalose from glucose-6-phosphate and glucose. The expression levels of the two trehalose phosphorylases associated with this pathway (AFUA_3G12100 and AFUA_ 5G14780) were also reduced in the DmybA mutant; the AFUA_5G14780 gene being one of the most down regulated genes (GFOLD 5 23.33). The down regula- tion of the genes involved in the synthesis of the treha- lose was in agreement with the lower amount of trehalose found in the conidium of the DmybA mutant ...
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... other conidial polyols varied differently from the trehalose in the DmybA, DvelC, DvelB, DveA, DvosA, DwetA and DcspA mutants (Fig. 7B). The DwetA and DcspA mutants had a very high amount of mannitol (>33 the concentration of the parental strain) whereas the DvosA, DvelB and DmybA mutants showed only a slight difference in the mannitol level with respect to the parental strain. DcspA, DwetA and DmybA mutants had a very low amount of arabitol whereas the DvosA and DvelB had an amount similar to the parental strain (Fig. 7C). These results suggest that the conidial intracellular osmolytes are controlled by compensatory pathways, which are differently activated in the different conidiating mutant ...
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... other conidial polyols varied differently from the trehalose in the DmybA, DvelC, DvelB, DveA, DvosA, DwetA and DcspA mutants (Fig. 7B). The DwetA and DcspA mutants had a very high amount of mannitol (>33 the concentration of the parental strain) whereas the DvosA, DvelB and DmybA mutants showed only a slight difference in the mannitol level with respect to the parental strain. DcspA, DwetA and DmybA mutants had a very low amount of arabitol whereas the DvosA and DvelB had an amount similar to the parental strain (Fig. 7C). These results suggest that the conidial intracellular osmolytes are controlled by compensatory pathways, which are differently activated in the different conidiating mutant ...
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Aspergillus fumigatus is an opportunistic human pathogen that causes various complications in patients with a weakened immune system functions. Asexual spores of A. fumigatus are responsible for initiation of aspergillosis. Long-term viability and proper germination of dormant conidia depend on trehalose accumulation, which protect the spores again...
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... In plants, Myb family proteins are predominantly known as transcription factors or repressors [18][19][20][21][22][23][24]. However, proteins that are not classical transcription factors can contain Myb DNA binding domains [23,[25][26][27][28][29][30][31][32][33]. In animal genomes, the Myb family is relatively small, consisting of only 4 to 5 proteins [13]. ...
The Myb family of transcription factors (TFs) is a large and functionally diverse group found in all eukaryotes, but its role in fungi remains poorly understood. Here, we characterized a Myb family TF called MoMyb13 in the rice blast fungus, Magnaporthe oryzae. MoMyb13 has two Myb domains and has orthologues only in ascomycete fungi. Localization experiments confirmed that MoMyb13 is located in the nuclei, as expected for a TF. Phenotypic analysis showed that MoMyb13 mutants exhibited reduced growth, whitened colony appearance and no conidia formation. Importantly, these mutants completely lost pathogenicity, despite being able to form appressoria at their hyphae ends, suggesting that these appressoria were non-functional for causing infection. Furthermore, the mutant colonies lost hydrophobicity and had significantly reduced expression of the hydrophobin MPG1. However, overexpression of MPG1 in the mutants restored their hydrophobicity, but not pathogenicity. Stress assay showed that the mutants were more sensitive to SDS, CR and H2O2, but more tolerant to NaCl and SOR. In summary, our study revealed the essential role of the MoMyb13 in the growth, conidiation, hydrophobicity, stress response and pathogenicity of M. oryzae, and provides valuable insights into the working mechanism of Myb TFs in fungi.
... GFP-Trap™ has been successfully used to identify protein complexes and modifications in fungi. It has been employed to identify complexes such as KERS, STRIPAK, RcLS2F, MAPK, Class V Myosin, CreA, and its phosphorylation, septin AspE phosphorylation, calcineurin binding protein phosphorylation, HOG MAPK complexes (SakA-MpkC), polo-like kinase PlkA interactions, Rho1-Rom2 complex, SCF complexes (F-box proteins), MybA interacting proteins and many others in Aspergilli [32,[52][53][54][55][56][57][58]. GFP-Trap™ has also been employed in other filamentous fungi, including N. crassa, enabling the identification of key components such as the exocyst complex, STRIPAK complex, septation initiation network (SIN) interactions, GUL-1 interacting proteins, and the VE-1/VE-2/LAE-1 complex [59][60][61][62]. ...
Fungi are a diverse group of organisms that can be both beneficial and harmful to mankind. They have advantages such as producing food processing enzymes and antibiotics, but they can also be pathogens and produce mycotoxins that contaminate food. Over the past two decades, there have been significant advancements in methods for studying fungal molecular biology. These advancements have led to important discoveries in fungal development, physiology, pathogenicity, biotechnology, and natural product research. Protein complexes and protein-protein interactions (PPIs) play crucial roles in fungal biology. Various methods, including yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), are used to investigate PPIs. However, affinity-based PPI methods like co-immunoprecipitation (Co-IP) are highly preferred because they represent the natural conditions of PPIs. In recent years, the integration of liquid chromatography coupled with mass spectrometry (LC-MS/MS) has been used to analyse Co-IPs, leading to the discovery of important protein complexes in filamentous fungi. In this review, we discuss the tandem affinity purification (TAP) method and single affinity purification methods such as GFP, HA, FLAG, and MYC tag purifications. These techniques are used to identify PPIs and protein complexes in filamentous fungi. Additionally, we compare the efficiency, time requirements, and material usage of Sepharose™ and magnetic-based purification systems. Overall, the advancements in fungal molecular biology techniques have provided valuable insights into the complex interactions and functions of proteins in fungi. The methods discussed in this review offer powerful tools for studying fungal biology and will contribute to further discoveries in this field.
... The role of MYB TFs in fungal growth and development, particularly in macro-fungi, is becoming the focus of research. In fungi, AfMybA, MYT1, and MYT2 have been found to be involved in growth and development [32][33][34]. Among macro-fungi, F. velutiper and Ganoderma lucidum MYB TFs were found to be involved in growth and development [35,36]. ...
MYB transcription factors (TFs) have been extensively studied in plant abiotic stress responses and growth and development. However, the role of MYB TFs in the heat stress response and growth and development of Pleurotus ostreatus remains unclear. To investigate the function of PoMYB12, PoMYB15, and PoMYB20 TFs in P. ostreatus, mutant strains of PoMYB12, PoMYB15, and PoMYB20 were generated using RNA interference (RNAi) and overexpression (OE) techniques. The results indicated that the mycelia of OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains exhibited positive effects under heat stress at 32 °C, 36 °C, and 40 °C. Compared to wild-type strains, the OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains promoted the growth and development of P. ostreatus. These mutant strains also facilitated the recovery of growth and development of P. ostreatus after 24 h of 36 °C heat stress. In conclusion, the expression of PoMYB12 and PoMYB20 supports the mycelium’s response to heat stress and enhances the growth and development of P. ostreatus, whereas PoMYB15 produces the opposite effect.
... One of the fundamental functions of fungal photoreception is self-defense against the harmful effects of UV light. Fungal spores are densely packed with protective pigments and osmolytes which protect the next generation of fungi from stressors such as UV light and oxidative stress [31][32][33]. The second fundamental function of photoreception is possibly to find optimum conditions or time for spore dispersal [34,35]. ...
Organisms have developed different features to capture or sense sunlight. Vertebrates have evolved specialized organs (eyes) which contain a variety of photosensor cells that help them to see the light to aid orientation. Opsins are major photoreceptors found in the vertebrate eye. Fungi, with more than five million estimated members, represent an important clade of living organisms which have important functions for the sustainability of life on our planet. Light signalling regulates a range of developmental and metabolic processes including asexual sporulation, sexual fruit body formation, pigment and carotenoid production and even production of secondary metabolites. Fungi have adopted three groups of photoreceptors: (I) blue light receptors, White Collars, vivid, cryptochromes, blue F proteins and DNA photolyases, (II) red light sensors, phytochromes and (III) green light sensors and microbial rhodopsins. Most mechanistic data were elucidated on the roles of the White Collar Complex (WCC) and the phytochromes in the fungal kingdom. The WCC acts as both photoreceptor and transcription factor by binding to target genes, whereas the phytochrome initiates a cascade of signalling by using mitogen-activated protein kinases to elicit its cellular responses. Although the mechanism of photoreception has been studied in great detail, fungal photoreception has not been compared with vertebrate vision. Therefore, this review will mainly focus on mechanistic findings derived from two model organisms, namely Aspergillus nidulans and Neurospora crassa and comparison of some mechanisms with vertebrate vision. Our focus will be on the way light signalling is translated into changes in gene expression, which influences morphogenesis and metabolism in fungi.
... AFUA_7G04300 was also implicated in broader cell wall activities, as it was predicted to regulate AFUA_5G13090, a putative alpha-1,2-mannosyltransferase and cell wall biosynthesis gene (92). ...
Aspergillus fumigatus is a notorious pathogenic fungus responsible for various harmful, sometimes lethal, diseases known as aspergilloses. Understanding the gene regulatory networks that specify the expression programs underlying this fungus′ diverse phenotypes can shed mechanistic insight into its growth, development, and determinants of pathogenicity. We used eighteen RNA-seq datasets (seventeen publicly available and one previously unpublished) of Aspergillus fumigatus to construct a comprehensive gene regulatory network resource. Our resource, named GRAsp (Gene Regulation of Aspergillus fumigatus), was able to recapitulate known regulatory pathways such as response to hypoxia, iron and zinc homeostasis, and secondary metabolite synthesis. Further, GRAsp was experimentally validated in two cases: one in which GRAsp accurately identified an uncharacterized transcription factor negatively regulating the production of the virulence factor gliotoxin and another where GRAsp revealed the bZip protein, AtfA, as required for fungal responses to microbial signals known as lipo-chitooligosaccharides. Our work showcases the strength of using network-based approaches to generate new hypotheses about regulatory relationships in Aspergillus fumigatus. We also unveil an online, user-friendly version of GRAsp available to the Aspergillus research community.
... A. fumigatus [196,221] RlmA Transcription factor that regulates mycotoxin production in conidia, as well as cell wall remodeling and synthesis, in particular, chitin. ...
Germination of conidia is an essential process within the Aspergillus life cycle and plays a major role during the infection of hosts. Conidia are able to avoid detection by the majority of leukocytes when dormant. Germination can cause severe health problems, specifically in immunocompromised people. Aspergillosis is most often caused by Aspergillus fumigatus (A. fumigatus) and affects neutropenic patients, as well as people with cystic fibrosis (CF). These patients are often unable to effectively detect and clear the conidia or hyphae and can develop chronic non-invasive and/or invasive infections or allergic inflammatory responses. Current treatments with (tri)azoles can be very effective to combat a variety of fungal infections. However, resistance against current azoles has emerged and has been increasing since 1998. As a consequence, patients infected with resistant A. fumigatus have a reported mortality rate of 88% to 100%. Especially with the growing number of patients that harbor azole-resistant Aspergilli, novel antifungals could provide an alternative. Aspergilloses differ in defining characteristics, but germination of conidia is one of the few common denominators. By specifically targeting conidial germination with novel antifungals, early intervention might be possible. In this review, we propose several morphotypes to disrupt conidial germination, as well as potential targets. Hopefully, new antifungals against such targets could contribute to disturbing the ability of Aspergilli to germinate and grow, resulting in a decreased fungal burden on patients.
... Blocking trehalose accumulation in A. fumigatus reduces spore viability and seriously impairs subsequent events, including germination, growth, and sporulation, after exposure to heat (50°C) (45). In A. fumigatus, the lack of trehalose in DmybA conidia results in the rapid loss of viability (46). Our results found that genes responsible for intracellular trehalose production were downregulated in DBbcmr1, and the content of intracellular trehalose was also reduced. ...
Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation.
... The only evidence of pseudogenization among the genetic determinants of virulence in the reference strains was observed in mybA (AFUA_3G07070), a transcription factor involved in conidiation and conidial viability (60), in strain A1163. MybA null mutants have reduced virulence compared to wild-type strains (60). ...
The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), first described in Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as opportunistic fungal pathogens from the genus Aspergillus. To gain insight into COVID-19-associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit significant differences from the genome of the Af293 reference strain. By examining a number of factors, including virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, secondary metabolite biosynthesis, and the MIC of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss-of-function mutations in genes known to increase virulence when deleted. Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains Af293 and CEA17, but similarly virulent to two other clinical strains of A. fumigatus. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA. IMPORTANCE The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has already killed millions of people. COVID-19 patient outcome can be further complicated by secondary infections, such as COVID-19-associated pulmonary aspergillosis (CAPA). CAPA is caused by Aspergillus fungal pathogens, but there is little information about the genomic and phenotypic characteristics of CAPA isolates. We conducted genome sequencing and extensive phenotyping of four CAPA isolates of Aspergillus fumigatus from Germany. We found that CAPA isolates were often, but not always, similar to other reference strains of A. fumigatus across 206 genetic determinants of infection-relevant phenotypes, including virulence. For example, CAPA isolate D was more virulent than other CAPA isolates and reference strains in an invertebrate model of fungal disease, but similarly virulent to two other clinical strains. These results expand our understanding of COVID-19-associated pulmonary aspergillosis.
... Abundant evidence in plants has confirmed the diverse roles of MYB genes in signal transduction, developmental regulation, biotic and abiotic stress responses, disease resistance, and secondary metabolism regulation (An et al. 2019; Lee & Seo 2019;Qing et al. 2019;Wu et al. 2019b;He et al. 2020;Ohno et al. 2020). Similarly, a few studies have shown that MYB genes participate in stress response and developmental regulation in fungi (Valsecchi et al. 2017;Wang et al. 2018;. ...
Numerous studies in plants have shown the vital roles of MYB transcription factors in signal transduction, developmental regulation, biotic/abiotic stress responses and secondary metabolism regulation. However, less is known about the functions of MYBs in Ganoderma . In this study, five medicinal macrofungi of genus Ganoderma were subjected to a genome-wide comparative analysis of MYB genes. A total of 75 MYB genes were identified and classified into four types: 1R-MYBs (52), 2R-MYBs (19), 3R-MYBs (2) and 4R-MYBs (2). Gene structure analysis revealed varying exon numbers (3-14) and intron lengths (7-1058 bp), and noncanonical GC-AG introns were detected in G. lucidum and G. sinense . In a phylogenetic analysis, 69 out of 75 MYB genes were clustered into 15 subgroups, and both single-copy orthologous genes and duplicated genes were identified. The promoters of the MYB genes harboured multiple cis-elements, and specific genes were co-expressed with the G. lucidum MYB genes, indicating the potential roles of these MYB genes in stress response, development and metabolism. This comprehensive and systematic study of MYB family members provides a reference and solid foundation for further functional analysis of MYB genes in Ganoderma species.
... The expression ratios were relative to TEF1 gene. Values are taken from the RNA-seq data analysis of Valsecchi et al. (2017). For this RNA-sequencing experiment, the fungus was grown for seven days on malt agar at 37 • C at room temperature. ...
Deacetylation of chitin by chitin deacetylases (Cda) results in the formation of chitosan. Chitosan, a polymer of β1,4 linked glucosamine, plays multiple roles in the function of the fungal cell wall, including virulence and evasion of host immune responses. In this study, the roles of chitosan and putative CDAs in cell wall structure and virulence of Aspergillus fumigatus were investigated. Low levels of chitosan were found in the conidial and cell wall of A. fumigatus. Seven putative CDA genes were identified, disrupted and the phenotype of the single mutants and the septuple mutants were investigated. No alterations in fungal cell wall chitosan levels, changes in fungal growth or alterations in virulence were detected in the single or septuple Δcda1-7 mutant strains. Collectively, these results suggest that chitosan is a minority component of the A. fumigatus cell wall, and that the seven candidate Cda proteins do not play major roles in fungal cell wall synthesis or virulence. However, Cda2 is involved in conidiation, suggesting that this enzyme may play a role in N-acetyl-glucosamine metabolism.
