Opportunistic Human Pathogenic Fungus Aspergillus Research Articles

The MYST Family Histone Acetyltransferase SasC Governs Diverse Biological Processes in Aspergillus fumigatus.

The conserved MYST proteins form the largest family of histone acetyltransferases (HATs) that acetylate lysines within the N-terminal tails of histone, enabling active gene transcription. Here, we have investigated the biological and regulatory functions of the MYST family HAT SasC in the opportunistic human pathogenic fungus Aspergillus fumigatus using a series of genetic, biochemical, pathogenic, and transcriptomic analyses. The deletion (Δ) of sasC results in a drastically reduced colony growth, asexual development, spore germination, response to stresses, and the fungal virulence. Genome-wide expression analyses have revealed that the ΔsasC mutant showed 2402 significant differentially expressed genes: 1147 upregulated and 1255 downregulated. The representative upregulated gene resulting from ΔsasC is hacA, predicted to encode a bZIP transcription factor, whereas the UV-endonuclease UVE-1 was significantly downregulated by ΔsasC. Furthermore, our Western blot analyses suggest that SasC likely catalyzes the acetylation of H3K9, K3K14, and H3K29 in A. fumigatus. In conclusion, SasC is associated with diverse biological processes and can be a potential target for controlling pathogenic fungi.

The human fungal pathogen Aspergillus fumigatus can produce the highest known number of meiotic crossovers.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Functional Characterization of the GNAT Family Histone Acetyltransferase Elp3 and GcnE in Aspergillus fumigatus.

Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a pivotal role in the regulation of gene expression and diverse biological processes. However, the function of GNAT family HATs, especially Elp3, in the opportunistic human pathogenic fungus Aspergillus fumigatus is largely unknown. To investigate the roles of the GNAT family HATs Elp3 and GcnE in the A. fumigatus, we have generated and characterized individual null Δelp3 and ΔgcnE mutants. The radial growth of fungal colonies was significantly decreased by the loss of elp3 or gcnE, and the number of asexual spores (conidia) in the ΔgcnE mutant was significantly reduced. Moreover, the mRNA levels of the key asexual development regulators were also significantly low in the ΔgcnE mutant compared to wild type (WT). Whereas both the Δelp3 and ΔgcnE mutants were markedly impaired in the formation of adherent biofilms, the ΔgcnE mutant showed a complete loss of surface structure and of intercellular matrix. The ΔgcnE mutant responded differently to oxidative stressors and showed significant susceptibility to triazole antifungal agents. Furthermore, Elp3 and GcnE function oppositely in the production of secondary metabolites, and the ΔgcnE mutant showed attenuated virulence. In conclusion, Elp3 and GcnE are associated with diverse biological processes and can be potential targets for controlling the pathogenic fungus.

The Lysine Demethylases KdmA and KdmB Differently Regulate Asexual Development, Stress Response, and Virulence in Aspergillus fumigatus.

Histone demethylases govern diverse cellular processes, including growth, development, and secondary metabolism. In the present study, we investigated the functions of two lysine demethylases, KdmA and KdmB, in the opportunistic human pathogenic fungus Aspergillus fumigatus. Experiments with mutants harboring deletions of genes encoding KdmA (ΔkdmA) and KdmB (ΔkdmB) showed that KdmA is necessary for normal growth and proper conidiation, whereas KdmB negatively regulates vegetative growth and conidiation. In both mutant strains, tolerance to H2O2 was significantly decreased, and the activities of both conidia-specific catalase (CatA) and mycelia-specific catalase (Cat1) were decreased. Both mutants had significantly increased sensitivity to the guanine nucleotide synthesis inhibitor 6-azauracil (6AU). The ΔkdmA mutant produced more gliotoxin (GT), but the virulence was not changed significantly in immunocompromised mice. In contrast, the production of GT and virulence were markedly reduced by the loss of kdmB. Comparative transcriptomic analyses revealed that the expression levels of developmental process-related genes and antioxidant activity-related genes were downregulated in both mutants. Taken together, we concluded that KdmA and KdmB have opposite roles in vegetative growth, asexual sporulation, and GT production. However, the two proteins were equally important for the development of resistance to 6AU.

The Putative APSES Transcription Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence in Aspergillus fumigatus.

The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA, brlA, and wetA were decreased in the ΔrgdA mutant compared to those in the wild type (WT). Moreover, ΔrgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the ΔrgdA mutant. Comparative transcriptomic analyses revealed that the ΔrgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the ΔrgdA mutant exhibited elevated production of GT, ΔrgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the ΔrgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling.IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.

Molecular Characterization of a Debilitation-Associated Partitivirus Infecting the Pathogenic Fungus Aspergillus flavus.

The opportunistic human pathogenic fungus Aspergillus flavus is known to be infected with mycoviruses. In this study, we report a novel mycovirus A. flavus partitivirus 1 (AfPV1) that was originally isolated from the abnormal colonial morphology isolate LD-3-8 of A. flavus. AfPV1 has spherical virus-like particles about 40 nm in diameter, and three double-stranded RNA (dsRNA) segments (dsRNA1, 2, and 3 with lengths of 1.7, 1.4, and 1.1 kbp, respectively) were packaged in the virions. dsRNA1, dsRNA2, and dsRNA3 each contained a single open reading frame and potentially encoded 62, 42, and 32 kDa proteins, respectively. The dsRNA1 encoded protein shows similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, and the dsRNA2 product has no significant similarity to any other capsid protein (CP) in the GenBank databases, beside some homology with the hypothetical “capsid” protein of a few partitiviruses. The dsRNA3 encodes a protein with no similarity to any protein in the GenBank database. SDS-PAGE and polypeptide mass fingerprint-mass spectrum (PMF-MS) analyses indicated that the CP of the AfPV1 was encoded by dsRNA2. Phylogenetic analysis showed that the AfPV1 and relative viruses were found in an unclassified group inside the Partitiviridae family. AfPV1 seems to result in debilitation symptoms, but had no significant effects to murine pathogenicity. These findings provide new insights into the partitiviruses taxonomy and the interactions between viruses and A. flavus.

Characteristics of a Regulator of G-Protein Signaling (RGS) rgsC in Aspergillus fumigatus.

The regulator of G-protein signaling (RGS) proteins have a conserved RGS domain that facilitates the intrinsic GTPase activity of an activated Gα subunit of heterotrimeric G protein, thereby attenuating signal transduction. Among six predicted RGS proteins in the opportunistic human pathogenic fungus Aspergillus fumigatus, only three (FlbA, GprK, and Rax1) have been studied. The unexplored RgsC composed of the Phox-associated (PXA), RGS, Phox homology (PX), and Nexin_C superfamily domains is highly conserved in many ascomycete fungi, suggesting a crucial role of RgsC in fungal biology. To address this, we have investigated functions of the rgsC gene. The deletion (Δ) of rgsC causes impaired vegetative growth and asexual development coupled with reduced expression of key developmental regulators. Moreover, ΔrgsC results in accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source. Furthermore, ΔrgsC causes reduced conidial tolerance to oxidative stress. In addition, activities and expression of catalases and superoxide dismutases (SODs) are severely decreased in the ΔrgsC mutant. The deletion of rgsC results in a slight reduction in conidial tolerance to cell wall damaging agents, yet significantly lowered mRNA levels of cell wall integrity/biogenesis transcription factors, indicating that RgsC may function in proper activation of cell wall stress response. The ΔrgsC mutant exhibits defective gliotoxin (GT) production and decreased virulence in the wax moth larvae, Galleria mellonella. Transcriptomic studies reveal that a majority of transporters is down-regulated by ΔrgsC and growth of the ΔrgsC mutant is reduced on inorganic and simple nitrogen medium, suggesting that RgsC may function in external nitrogen source sensing and/or transport. In summary, RgsC is necessary for proper growth, development, stress response, GT production, and external nutrients sensing.

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus.

Expression and Activity of Catalases Is Differentially Affected by GpaA (Ga) and FlbA (Regulator of G Protein Signaling) in Aspergillus fumigatus

Vegetative growth signaling of the opportunistic human pathogenic fungus Aspergillus fumigatus is mediated by GpaA (Gα). FlbA is a regulator of G protein signaling, which attenuates GpaA-mediated growth signaling in this fungus. The flbA deletion (ΔflbA) and the constitutively active GpaA (GpaAQ204L) mutants exhibit enhanced proliferation, precocious autolysis, and reduced asexual sporulation. In this study, we demonstrate that both mutants also show enhanced tolerance against H2O2 and their radial growth was approximately 1.6 fold higher than that of wild type (WT) in medium with 10 mM H2O2. We performed quantitative PCR (qRT-PCR) for examination of mRNA levels of three catalase encoding genes (catA, cat1, and cat2) in WT and the two mutants. According to the results, while levels of spore-specific catA mRNA were comparable among the three strains, cat1 and cat2 mRNA levels were significantly higher in the two mutants than in WT. In particular, the ΔflbA mutant showed significantly enhanced and prolonged expression of cat1 and precocious expression of cat2. In accordance with this result, activity of the Cat1 protein in the ΔflbA mutant was higher than that of gpaAQ204L and WT strains. For activity of the Cat2 protein, both mutants began to show enhanced activity at 48 and 72 hr of growth compared to WT. These results lead to the conclusion that GpaA activates expression and activity of cat1 and cat2, whereas FlbA plays an antagonistic role in control of catalases, leading to balanced responses to neutralizing the toxicity of reactive oxygen species.

Aspergillus fumigatus melanins: interference with the host endocytosis pathway and impact on virulence

The opportunistic human pathogenic fungus Aspergillus fumigatus produces at least two types of melanin, namely pyomelanin and dihydroxynaphthalene (DHN) melanin. Pyomelanin is produced during tyrosine catabolism via accumulation of homogentisic acid. Although pyomelanin protects the fungus against reactive oxygen species (ROS) and acts as a defense compound in response to cell wall stress, mutants deficient for pyomelanin biosynthesis do not differ in virulence when tested in a murine infection model for invasive pulmonary aspergillosis. DHN melanin is responsible for the characteristic gray-greenish color of A. fumigatus conidia. Mutants lacking a functional polyketide synthase PksP, the enzyme responsible for the initial step in DHN-melanin formation, i.e., the synthesis of naphthopyrone, produce white spores and are attenuated in virulence. The activity of PksP was found to be essential not only for inhibition of apoptosis of phagocytes by interfering with the host PI3K/Akt signaling cascade but also for effective inhibition of acidification of conidia-containing phagolysosomes. These features allow A. fumigatus to survive in phagocytes and thereby to escape from human immune effector cells and to become a successful pathogen.

Pyomelanin formation in Aspergillus fumigatus requires HmgX and the transcriptional activator HmgR but is dispensable for virulence.

The opportunistic human pathogenic fungus Aspergillus fumigatus is able to produce the dark brown pigment pyomelanin by degradation of L-tyrosine. Pyomelanin was shown to protect the fungus against reactive oxygen intermediates as well as cell wall disturbing compounds and is therefore assumed to protect against immune effector cells during the infection process. Several genes for tyrosine degradation and pyomelanin formation are organized in a cluster in the genome of A. fumigatus. Here, we aimed at further analyzing tyrosine degradation and a possible role of pyomelanin in virulence. For this purpose, the function of two not yet characterized genes of the cluster, i.e., hmgX and hmgR, was analyzed. Generation of corresponding gene deletion mutants and reconstituted strains revealed that hmgX and hmgR are essential for tyrosine degradation. Both mutants, ΔhmgX and ΔhmgR, were not able to use tyrosine as sole carbon or nitrogen source and revealed impaired pyomelanin production. HmgR harbors a Zn(II)2Cys6-DNA binding domain. Analyses of the steady state mRNA levels revealed that HmgR acts as a transcriptional activator for the genes of the tyrosine degradation cluster. Consistently, an HmgR-eGFP fusion protein was localized in the nucleus of A. fumigatus cells. By contrast, HmgX was found to be localized in the cytoplasm and does not contribute to regulation of gene transcription. HPLC analyses showed that HmgX is crucial for the conversion of p-hydroxyphenylpyruvate to homogentisic acid, the main intermediate in pyomelanin formation. Thus, HmgX is supposed to function as an accessory factor to mediate specific activity of HppD. Remarkably, the ability to degrade tyrosine and to form pyomelanin is dispensable for virulence of A. fumigatus in a murine infection model.

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.

Secreted Aspergillus fumigatus protease Alp1 degrades human complement proteins C3, C4, and C5.

The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack.

The Opportunistic Human Pathogenic FungusAspergillus fumigatusEvades the Host Complement System

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.

Apoptosis inhibition of alveolar macrophages upon interaction with conidia ofAspergillus fumigatus

The opportunistic human pathogenic fungus Aspergillus fumigatus (Af) causes the majority of cases of invasive aspergillosis. Because Af enters the human body through inhalation of airborne conidia, the interaction of conidia with the innate immune system (alveolar macrophages) plays a key role in the etiology of aspergillosis. Therefore, it is of central interest to investigate response mechanisms of alveolar macrophages upon interaction with Af. Here, it is demonstrate that Af inhibited host cell apoptosis of alveolar macrophages, one of the major defense immune effector cells against this pathogen. This unexpected result was due to inhibition of caspase 3 by a yet unknown mechanism.

Methylcitrate synthase from Aspergillus fumigatus

Methylcitrate synthase is a key enzyme of the methylcitrate cycle and required for fungal propionate degradation. Propionate not only serves as a carbon source, but also acts as a food preservative (E280-283) and possesses a negative effect on polyketide synthesis. To investigate propionate metabolism from the opportunistic human pathogenic fungus Aspergillus fumigatus, methylcitrate synthase was purified to homogeneity and characterized. The purified enzyme displayed both, citrate and methylcitrate synthase activity and showed similar characteristics to the corresponding enzyme from Aspergillus nidulans. The coding region of the A. fumigatus enzyme was identified and a deletion strain was constructed for phenotypic analysis. The deletion resulted in an inability to grow on propionate as the sole carbon source. A strong reduction of growth rate and spore colour formation on media containing both, glucose and propionate was observed, which was coincident with an accumulation of propionyl-CoA. Similarly, the use of valine, isoleucine and methionine as nitrogen sources, which yield propionyl-CoA upon degradation, inhibited growth and polyketide production. These effects are due to a direct inhibition of the pyruvate dehydrogenase complex and blockage of polyketide synthesis by propionyl-CoA. The surface of conidia was studied by electron scanning microscopy and revealed a correlation between spore colour and ornamentation of the conidial surface. In addition, a methylcitrate synthase deletion led to an attenuation of virulence, when tested in an insect infection model and attenuation was even more pronounced, when whitish conidia from glucose/propionate medium were applied. Therefore, an impact of methylcitrate synthase in the infection process is discussed.

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.