Transcription factor CreA is involved in the inverse regulation of biofilm formation and asexual development through distinct pathways in Aspergillus fumigatus.

The exopolysaccharide galactosaminogalactan (GAG) contributes to biofilm formation and virulence in the pathogenic fungus Aspergillus fumigatus. Increasing evidence indicates that GAG production is inversely linked with asexual development. However, the mechanisms underlying this regulatory relationship are unclear. In this study, we found that the dysfunction of CreA, a conserved transcription factor involved in carbon catabolite repression in many fungal species, causes abnormal asexual development (conidiation) under liquid-submerged culture conditions specifically in the presence of glucose. The loss of creA decreased GAG production independent of carbon sources. Furthermore, CreA contributed to asexual development and GAG production via distinct pathways. CreA promoted A. fumigatus GAG production by positively regulating GAG biosynthetic genes (uge3 and agd3). CreA suppressed asexual development in glucose liquid-submerged culture conditions via central conidiation genes (brlA, abaA, and wetA) and their upstream activators (flbC and flbD). Restoration of brlA expression to the wild-type level by flbC or flbD deletion abolished the abnormal submerged conidiation in the creA null mutant but did not restore GAG production. The C-terminal region of CreA was crucial for the suppression of asexual development, and the repressive domain contributed to GAG production. Overall, CreA is involved in GAG production and asexual development in an inverse manner.

Transcription factors SltA and CrzA reversely regulate calcium homeostasis under calcium-limited conditions.

IMPORTANCE: Calcium ions are ubiquitous intracellular signaling molecules for many signaling pathways regulating the fungal response to stress and antifungal drugs. The concentration of intracellular calcium is tightly regulated in its storage, release, and distribution. CrzA is the best-studied transcription factor that regulates this process under sufficient calcium or other external signals. However, CrzA was excluded from nuclei and then lost transcriptional activation under calcium-limited conditions. The regulators in the Ca2+ signaling pathway under calcium-limited conditions remain unclear. Here, we identified SltA as a key regulator in the Ca2+ signaling pathway under calcium-limited conditions, and the underlying mechanisms were further explored in Aspergillus fumigatus. These findings reveal a transcriptional control pathway that precisely regulates calcium homeostasis under calcium-limited conditions.

Spt20, a Structural Subunit of the SAGA Complex, Regulates Aspergillus fumigatus Biofilm Formation, Asexual Development, and Virulence.

The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. Previous work showed that in A. fumigatus, the Lim domain-binding protein PtaB can form a complex with the sequence-specific transcription factor SomA for regulating GAG biosynthesis, biofilm formation, and asexual development. However, transcriptional coactivators required for biofilm formation in A. fumigatus remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in the GAG biosynthesis, biofilm formation, conidiation, and virulence of A. fumigatus. RNA sequence data demonstrated that Spt20 positively regulates the expression of the GAG biosynthesis genes uge3 and agd3, the developmental regulator medA, and genes involved in the conidiation pathway. Moreover, more than 10 subunits of the SAGA complex (known from yeast) could be immunoprecipitated with Spt20, suggesting that Spt20 acts as a structural subunit of the SAGA complex. Furthermore, distinct modules of SAGA regulate GAG biosynthesis, biofilm formation, and asexual development in A. fumigatus to various degrees. In summary, the novel biofilm regulator Spt20 is reported, which plays a crucial role in the regulation of fungal asexual development, GAG biosynthesis, and virulence in A. fumigatus. These findings expand knowledge on the regulatory circuits of the SAGA complex relevant for the biofilm formation and asexual development of A. fumigatus. IMPORTANCE Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA-binding factors to transcriptional coactivators (chromatin regulators and the general transcription machinery) and their regulators. Previous research indicated that the sequence-specific complex SomA/PtaB regulates the biofilm formation and asexual development of Aspergillus fumigatus. However, transcriptional coactivators working with sequence-specific transcription factors to regulate A. fumigatus biofilm formation remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a novel regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in galactosaminogalactan (GAG) production, conidiation, and virulence. Moreover, nearly all modules of the SAGA complex were required for the biofilm formation and asexual development of A. fumigatus. These results establish the SAGA complex as a transcriptional coactivator required for the biofilm formation and asexual development of A. fumigatus.

The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

PtaB, a lim-domain binding protein in Aspergillus fumigatus regulates biofilm formation and conidiation through distinct pathways.

The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. The developmental modifiers MedA, StuA, and SomA regulate GAG biosynthesis, but the mechanisms underlying this regulation are poorly understood. PtaB is a lim-domain binding protein that interacts with the transcription factor SomA and is required for normal conidiation and biofilm formation. Disruption of ptaB resulted in impaired GAG production and conidiation in association with a markedly reduced expression of GAG biosynthetic genes (uge3 and agd3), developmental regulators (medA and stuA), and genes involved in the core conidiation pathway. Overexpression of medA and dual overexpression of uge3 and agd3 in the ΔptaB mutant increased biofilm formation but not conidiation, whereas overexpression of core conidiation genes rescued conidiation but not biofilm formation. Overexpression of stuA modestly increased both conidiation and biofilm formation. Analysis of ptaB truncation mutants revealed that overexpression of the lim-domain binding region restored conidiation but not biofilm formation, suggesting that ptaB may govern these processes by interacting with different partners. These studies establish that PtaB governs GAG biosynthesis at the level of substrate availability and polymer deacetylation and that PtaB-mediated biofilm formation and conidiation are largely independent pathways.

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS). The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs), a putative proton V-type proton ATPase (Vma5 homolog) and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans.

The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans.

The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans Additionally, the hypersensitivity of the ΔcnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca(2+) concentration ([Ca(2+)]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca(2+)]c, but a deficiency in both cnaA and cchA switches the [Ca(2+)]c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the ΔcnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the ΔcnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7:e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca(2+)]c Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca(2+) homeostasis in response to different environments.

Calcium signaling mediates antifungal activity of triazole drugs in the Aspergilli.

Azoles are widely applied and largely effective as antifungals; however, the increasing prevalence of clinically resistant isolates has yet to be matched by approaches to improve the efficacy of antimicrobial therapy. In this study, using the model fungus Aspergillus nidulans and one of the most common human pathogen Aspergillus fumigatus as research materials, we present the evidence that calcium signaling is involved in the azole-antifungals-induced stress-response reactions. In normal media, antifungal-itraconazole (ITZ) is able to induce the [Ca(2+)]c increased sharply but the addition of calcium chelator-EGTA or BAPTA almost blocks the calcium influx responses, resulted in the dramatically decreasing of [Ca(2+)]c transient. Real-time PCR analysis verified that six-tested Ca(2+)-inducible genes-two calcium channels (cchA/midA), a calmodulin-dependent phosphatase-calcineurin (cnaA), a transcription factor-crzA, and two calcium transporters (pmrA/pmcA)-could be transiently up-regulated by adding ITZ, indicating these components are involved in the azole stress-response reaction. Defect of cnaA or crzA caused more susceptibility to azole antifungals than did single mutants or double deletions of midA and cchA. Notably, EGTA may influence Rh123 accumulation as an azole-mimicking substrate through the process of the drug absorption. In vivo studies of a Galleria mellonella model identified that the calcium chelator works as an adjunct antifungal agent with azoles for invasive aspergillosis. Most importantly, combination of ITZ and EGTA or ITZ with calcium signaling inhibitor-FK506 greatly enhances the ITZ efficacy. Thus, our study provides potential clues that specific inhibitors of calcium signaling could be clinically useful adjuncts to conventional azole antifungals in the Aspergilli.

FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

Riboflavin level manipulates the successive developmental sequences in Aspergillus nidulans.

Auxotrophic markers are useful in fungal genetic analysis. Among the auxotrophic markers, riboB2 is one of the most commonly used markers in many laboratory strains. However, riboB2 mutants in Aspergillus nidulans confer self-sterility and thus are unable to form hybrid cleistothecia by outcross when both parent strains harbor riboB2 auxotrophic marker under the standard protocol. To assess the role of riboflavin during the different developmental stages of A. nidulans, the limited concentrations of riboflavin were monitored. The commonly used dosage of riboflavin (2.5 µg/ml) in the standard medium recipe is enough for hyphal growth and conidiation in the riboflavin auxotrophic riboB2 mutants (enough at 0.02 and 0.5 µg/ml, respectively) in A. nidulans. However, the dosage is not enough to support mature cleistothecium formation. Furthermore, the self-sterile defects in riboB2 mutants on standard medium could be restored by the addition of 25 µg/ml riboflavin, although the required riboflavin concentrations are varied in different genotype strains in A. nidulans. Most importantly, the outcross between riboB2 mutants could also be achieved by the supply of riboflavin in the sexual developmental stage. Our results highlight the potential roles of auxotrophic markers in the development of fungi and improve the efficiency of the genetic analysis in A. nidulans.

Putative PmrA and PmcA are important for normal growth, morphogenesis and cell wall integrity, but not for viability in Aspergillus nidulans.

P-type Ca(2+)-transporting ATPases are Ca(2+) pumps, extruding cytosolic Ca(2+) to the extracellular environment or the intracellular Ca(2+) store lumens. In budding yeast, Pmr1 (plasma membrane ATPase related), and Pmc1 (plasma membrane calcium-ATPase) cannot be deleted simultaneously for it to survive in standard medium. Here, we deleted two putative Ca(2+) pumps, designated AnPmrA and AnPmcA, from Aspergillus nidulans, and obtained the mutants ΔanpmrA and ΔanpmcA, respectively. Then, using ΔanpmrA as the starting strain, the promoter of its anpmcA was replaced with the alcA promoter to secure the mutant ΔanpmrAalcApmcA or its anpmcA was deleted completely to produce the mutant ΔanpmrAΔpmcA. Different from the case in Saccharomyces cerevisiae, double deletion of anpmrA and anpmcA was not lethal in A. nidulans. In addition, deletion of anpmrA and/or anpmcA had produced growth defects, although overexpression of AnPmc1 in ΔanpmrAalcApmcA could not restore the growth defects that resulted from the loss of AnPmrA. Moreover, we found AnPmrA was indispensable for maintenance of normal morphogenesis, especially in low-Ca(2+)/Mn(2+) environments. Thus, our findings suggest AnPmrA and AnPmcA might play important roles in growth, morphogenesis and cell wall integrity in A. nidulans in a different way from that in yeasts.

The EH domain-containing protein, EdeA, is involved in endocytosis, cell wall integrity, and pathogenicity in Aspergillus fumigatus.

Endocytosis has been extensively studied in yeasts, where it plays crucial roles in growth, signaling regulation, and cell-surface receptor internalization. However, the biological functions of endocytosis in pathogenic filamentous fungi remain largely unexplored. In this study, we aimed to functionally characterize the roles of EdeA, an ortholog of the Saccharomyces cerevisiae endocytic protein Ede1, in Aspergillus fumigatus. EdeA was observed to be distributed as patches on the plasma membrane and concentrated in the subapical collar of hyphae, a localization characteristic of endocytic proteins. Loss of edeA caused defective hyphal polarity, reduced conidial production, and fewer sites of endocytosis initiations than that of the parental wild type. Notably, the edeA null mutant exhibited increased sensitivity to cell wall-disrupting agents, indicating a role for EdeA in maintaining cell wall integrity in A. fumigatus. This observation was further supported by the evidence showing that the thickness of the cell wall in the ΔedeA mutant increased, accompanied by abnormal activation of MpkA, a key component in the cell wall integrity pathway. Additionally, the ΔedeA mutant displayed increased pathogenicity in the Galleria mellonella wax moth infection model, possibly due to alterations in cell wall morphology. Site-directed mutagenesis identified the conserved residue E348 within the third EH (Eps15 homology) domain of EdeA as crucial for its subcellular localization and functions. In conclusion, our results highlight the involvement of EdeA in endocytosis, hyphal polarity, cell wall integrity, and pathogenicity in A. fumigatus. IMPORTANCE: Aspergillus fumigatus is a significant human pathogenic fungus known to cause invasive aspergillosis, a disease with a high mortality rate. Understanding the basic principles of A. fumigatus pathogenicity is crucial for developing effective strategies against this pathogen. Previous research has underscored the importance of endocytosis in the infection capacity of pathogenic yeasts; however, its biological function in pathogenic mold remains largely unexplored. Our characterization of EdeA in A. fumigatus sheds light on the role of endocytosis in the development, stress response, and pathogenicity of pathogenic molds. These findings suggest that the components of the endocytosis process may serve as potential targets for antifungal therapy.

CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in entomopathogenic fungus Beauveria bassiana.

Infection of insects by the entomopathogenic fungus Beauveria bassiana proceeds via attachment and penetration of the host cuticle. The outermost epicuticular layer or waxy layer of the insect represents a structure rich in lipids including abundant amounts of hydrocarbons and fatty acids. A member of a novel cytochrome P450 subfamily, CYP52X1, implicated in fatty acid assimilation by B. bassiana was characterized. B. bassiana targeted gene knockouts lacking Bbcyp52x1 displayed reduced virulence when topically applied to Galleria mellonella, but no reduction in virulence was noted when the insect cuticle was bypassed using an intrahemoceol injection assay. No significant growth defects were noted in the mutant as compared with the wild-type parent on any lipids substrates tested including alkanes and fatty acids. Insect epicuticle germination assays, however, showed reduced germination of ΔBbcyp52x1 conidia on grasshopper wings as compared with the wild-type parent. Complementation of the gene-knock with the full-length gene restored virulence and insect epicuticle germination to wild-type levels. Heterologous expression of CYP52X1 in yeast was used to characterize the substrate specificity of the enzyme. CYP52X1 displayed the highest activity against midrange fatty acids (C12:0 and C14:0) and epoxy stearic acid, 4-8-fold lower activity against C16:0, C18:1, and C18:2, and little to no activity against C9:0 and C18:0. Analyses of the products of the C12:0 and C18:1 reactions confirmed NADPH-dependent regioselective addition of a terminal hydroxyl to the substrates (ω-hydroxylase). These data implicate CYP52X1 as contributing to the penetration of the host cuticle via facilitating the assimilation of insect epicuticle lipids.

Phosphoribosyl pyrophosphate synthetase, as a suppressor of the sepH mutation in Aspergillus nidulans, is required for the proper timing of septation.

Timely cytokinesis/septation is essential for hyphal growth and conidiation in Aspergillus nidulans. Genetic analyses have identified that A. nidulans has components of the septum initiation network (SIN) pathway; one of these, SEPH, is a key player for early events during cytokinesis. However, little is known about how the SEPH kinase cascade is regulated by other components. Here, we demonstrate that the phosphoribosyl pyrophosphate synthetase family acts antagonistically against the SIN so that the downregulation of AnPRS family can bypass the requirements of the SIN for septum formation and conidiation. The transcription defect of the Anprs gene family accompanied with the reduction of AnPRS activity causes the formation of hyper-septation as well as the restoration of septation and conidiation in the absence of SEPH. Clearly, the timing and positioning of septation is related to AnPRS activity. Moreover, with the extensive yeast two-hybrid analysis and rescue combination experiments, it demonstrated that AnPRS members are able to form the heterodimers for functional interacting entities but they appear to contribute so unequally that Anprs1 mutant display relatively normal septation, but Anprs2 deletion is lethal. Thus, compared to in yeast, the AnPRS family may have a unique regulation mechanism during septation in filamentous fungi.

Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development and pathogenesis in the entomopathogenic fungus, Beauveria bassiana.

The entomogenous filamentous fungus, Beauveria bassiana expresses two hydrophobin genes, hyd1 and hyd2, hypothesized to be involved in cell surface hydrophobicity, adhesion, virulence, and to constitute the protective spore coat structure known as the rodlet layer. Targeted gene inactivation of hyd1 resulted in seemingly 'bald' conidia that contained significantly altered surface fascicles or bundles. These cells displayed decreased spore hydrophobicity, loss of water mediated dispersal, changes in surface carbohydrate epitopes and ß-1,3-glucan distribution, lowered virulence in insect bioassays, but no effect on adhesion. In contrast, Δhyd2 mutants retained distorted surface bundles, but truncated/incomplete rodlets could be seen within the bundles. Δhyd2 conidia displayed both decreased cell surface hydrophobicity and adhesion, but the mutant was unaffected in virulence. The double Δhyd1Δhyd2 mutant was distinct from the single mutants, lacking both bundles and rodlets, and displaying additively decreased cell surface hydrophobicity, reduced cell attachment and lowered virulence than the Δhyd1 mutant. Epitope tagged constructs of the proteins were used to examine the expression and distribution of the proteins and to demonstrate the continued presence of Hyd2 in the Δhyd1 strain and vice versa. The implications of our results with respect to fascicle and rodlet assembly on the spore surface are discussed.

Filamentous fungal biofilms: Conserved and unique aspects of extracellular matrix composition, mechanisms of drug resistance and regulatory networks in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is an ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses, highlighting the importance of defining the mechanisms underlying biofilm development and associated emergent properties. A. fumigatus biofilms display a morphology and architecture that is distinct from bacterial and yeast biofilms. Moreover, A. fumigatus biofilms display unique characteristics in the composition of their extracellular matrix (ECM) and the regulatory networks governing biofilm formation. This review will discuss our current understanding of the form and function of A. fumigatus biofilms, including the unique components of ECM matrix, potential drug resistance mechanisms, the regulatory networks governing A. fumigatus biofilm formation, and potential therapeutics targeting these structures.

Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidylinositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and virulence.

Beauveria bassiana is a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes ß-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putative B. bassiana GPI-anchored ß-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized. B. bassiana targeted gene knockouts lacking Bbgas1 were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including ß-1,3-glucans, in the ΔBbgas1 strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1 conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1 conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1 knockout showed reduced virulence in Galleria mellonella insect bioassays in both topical and intrahemocoel-injection assays. B. bassiana ΔBbgas1 strains complemented with the complete Bbgas1 open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. The Bbgas1 gene did not complement the phenotype of a Saccharomyces cerevisiae ß-1,3-glucanosyltransferase Δgas1 mutant, indicating that this family of enzymes likely possess discrete cellular functions.

High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana displays a broad insect host range and serves as a model for examining host-pathogen interactions. Rapid construction and screening of random-insertion mutants of B. bassiana provides a powerful tool to dissect the molecular mechanisms of fungal virulence. LiAc/DMSO treated B. bassiana blastospores were found to be highly competent to transformation using linear DNA and a polyethylene glycol-based method. Selection on cellophane-layered Czapek-Dox agar at a lowered pH (from 7.5 to 6.3) greatly decreased background growth of non-transformed cells and improved screening of transformants. Optimization of the protocol using integration of the bar phosphinothricin resistance gene resulted in high transformation rates (200-250 transformants/µg DNA/10(8)cells). A collection of ∼ 4000 insertion mutants was examined via high-throughput screens for hydrocarbon utilization. One mutant was isolated that grew poorly on both n-hexadecane and tributyrin. The random insertion site was mapped to a gene that displayed homology to vitamin H (biotin)/tartrate transporters. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the mutant. This system provides a simple and rapid method for the generation and screening of insertion mutants and should expand our ability to genetically analyze the B. bassiana lifestyle.

A Transient Receptor Potential-like Calcium Ion Channel in the Filamentous Fungus Aspergillus nidulans.

Transient Receptor Potential (TRP) proteins constitute a superfamily that encodes transmembrane ion channels with highly diverse permeation and gating properties. Filamentous fungi possess putative TRP channel-encoded genes, but their functions remain elusive. Here, we report that a putative TRP-like calcium channel, trpR, in the filamentous fungus Aspergillus nidulans, performs important roles in conidiation and in adapting to cell wall disruption reagents in a high temperature-induced defect-dependent manner, especially under a calcium-limited culture condition. The genetic and functional relationship between TrpR and the previously identified high-affinity calcium channels CchA/MidA indicates that TrpR has an opposite response to CchA/MidA when reacting to cell wall disruption reagents and in regulating calcium transients. However, a considerable addition of calcium can rescue all the defects that occur in TrpR and CchA/MidA, meaning that calcium is able to bypass the necessary requirement. Nevertheless, the colocalization at the membrane of the Golgi for TrpR and the P-type Golgi Ca2+ ATPase PmrA suggests two channels that may work as ion transporters, transferring Ca2+ from the cytosol into the Golgi apparatus and maintaining cellular calcium homeostasis. Therefore, combined with data for the trpR deletion mutant revealing abnormal cell wall structures, TrpR works as a Golgi membrane calcium ion channel that involves cell wall integration.