Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.     

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.     

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion,Cell Wall Integrity,Biofilm Formation,and Virulence

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased β-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl₂, CaCl₂, and LiCl, but it is more resistant to ZnSO₄. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.     

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.     

Novel Mechanism Coupling Cyclic AMP-Protein Kinase A Signaling and Golgi Trafficking via Gyp1 Phosphorylation in Polarized Growth

The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1^4E) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1^4A) abolished it. Gyp1^4E exhibited enhanced association with the actin motor Myo2, while Gyp1^4A showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1^R292K) showed strong polarization similar to that seen with Gyp1^4E, indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.     

Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations.     

Divergent Protein Kinase A isoforms co-ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus

The genome of Aspergillus fumigatus encodes two isoforms of the catalytic subunit of the cAMP-dependent Protein Kinase (PKA). Although deletion of the class I isoform, pkaC1, leads to an attenuation of virulence, the function of the class II subunit, PkaC2, was previously uninvestigated. In this report, we demonstrate that both isoforms act in concert to support various physiologic processes that promote the virulence of this pathogen. Whereas pkaC1 and pkaC2 single-deletion mutants display wild-type conidial germination, a double-deletion mutant is delayed in germination in response to environmental nutrients. Furthermore, PkaC1 and PkaC2 interact to positively regulate flux through the carbohydrate catabolic pathway and, consequently, the ΔpkaC1ΔpkaC2 mutant is unable to grow on low glucose concentrations. Importantly, the reduced germinative capacity and inability to utilize glucose observed for the ΔpkaC1ΔpkaC2 strain correlated with an inability of the mutant to establish infection in a murine model. Conversely, overexpression of pkaC2 both promotes the in vitro growth on glucose, and restores the fungal burden and mortality associated with the ΔpkaC1 to that of the wild-type organism. Taken together, these data demonstrate the functional capacity of pkaC2 and emphasize the importance of PKA-mediated metabolic control in the pathogenic potential of A. fumigatus.     

Proteome Analysis of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Total Membrane Proteins Identifies Proteins Associated with the Glycoconjugates and Cell Wall Biosynthesis Using 2D LC-MS/MS

We attempted to identify membrane proteins associated with the glycoconjugates and cell wall biosynthesis in the total membrane preparations of Aspergillus fumigatus. The total membrane preparations were first run on 1D gels, and then the stained gels were cut and submitted to in-gel digestion followed by 2D LC-MS/MS and database search. A total of 530 proteins were identified with at least two peptides detected with MS/MS spectra. Seventeen integral membrane proteins were involved in N-, O-glycosylation or GPI anchor biosynthesis. Nine membrane proteins were involved in cell wall biosynthesis. Eight proteins were identified as enzymes involved in sphingolipid synthesis. In addition, the proteins involved in cell wall and ergosterol biosynthesis can potentially be used as antifungal drug targets. Our method, for the first time, clearly provided a global view of the membrane proteins associated with glycoconjugates and cell wall biosynthesis in the total membrane proteome of A. fumigatus.     

Discovery of novel pathways for carbohydrate metabolism

Closing the gap between the increasing availability of complete genome sequences and the discovery of novel enzymes in novel metabolic pathways is a significant challenge. Here, we review recent examples of assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized proteins, with a focus on enzymes and metabolic pathways involved in the catabolism and biosynthesis of monosaccharides and polysaccharides. The most effective approaches are based on analyses of sequence-function space in protein families that provide clues for the predictions of the functions of the uncharacterized enzymes. As summarized in this Opinion, this approach allows the discovery of the catabolism of new molecules, new pathways for common molecules, and new enzymatic chemistries.     

Dysregulated gliotoxin biosynthesis attenuates the production of unrelated biosynthetic gene cluster-encoded metabolites in Aspergillus fumigatus

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus. The gliotoxin bis-thiomethyltransferase, GtmA, attenuates gliotoxin biosynthesis in A. fumigatus by conversion of dithiol gliotoxin to bis-thiomethylgliotoxin (BmGT). Here we show that disruption of dithiol gliotoxin bis-thiomethylation functionality in A. fumigatus results in significant remodelling of the A. fumigatus secondary metabolome upon extended culture. RP-HPLC and LC–MS/MS analysis revealed the reduced production of a plethora of unrelated biosynthetic gene cluster-encoded metabolites, including pseurotin A, fumagillin, fumitremorgin C and tryprostatin B, occurs in A. fumigatus ΔgtmA upon extended incubation. Parallel quantitative proteomic analysis of A. fumigatus wild-type and ΔgtmA during extended culture revealed cognate abundance alteration of proteins encoded by relevant biosynthetic gene clusters, allied to multiple alterations in hypoxia-related proteins. The data presented herein reveal a previously concealed functionality of GtmA in facilitating the biosynthesis of other BGC-encoded metabolites produced by A. fumigatus.     

A型激酶锚定蛋白及其复合物结构与功能

A型激酶锚定蛋白(A-kinase anchoring proteins,AKAPs)是一类结构不同而功能相关的蛋白家族,其主要功能是将cAMP依赖性蛋白激酶A（PKA）锚定于特定的亚细胞结构.PKA是第二信使cAMP的主要效应器,而AKAPs在靶向定位和调节PKA介导的磷酸化事件方面扮演重要角色. AKAPs更为重要的功能是与多种信号分子形成信号复合物,从时间和空间上整合cAMP-PKA和其他信号途径.本文将对AKAPs及其信号复合物的结构特点和参与细胞信号转导的功能机制及其研究现状进行概述.     

The hypoxia‐induced dehydrogenase HorA is required for coenzyme Q10 biosynthesis,azole sensitivity and virulence of Aspergillus fumigatus

Aspergillus fumigatus is the predominant airborne pathogenic fungus causing invasive aspergillosis in immunocompromised patients. During infection A. fumigatus has to adapt to oxygen‐limiting conditions in inflammatory or necrotic tissue. Previously, we identified a mitochondrial protein to be highly up‐regulated during hypoxic adaptation. Here, this protein was found to represent the novel oxidoreductase HorA. In Saccharomyces cerevisiae a homologue was shown to play a role in biosynthesis of coenzyme Q. Consistently, reduced coenzyme Q content in the generated ΔhorA mutant indicated a respective function in A. fumigatus. Since coenzyme Q is involved in cellular respiration and maintaining cellular redox homeostasis, the strain ΔhorA displayed an impaired response to both oxidative and reductive stress, a delay in germination and an accumulation of NADH. Moreover, an increased resistance against antifungal drugs was observed. All phenotypes were completely reversed by the addition of the synthetic electron carrier menadione. The deletion strain ΔhorA showed significantly attenuated virulence in two murine infection models of invasive pulmonary aspergillosis. Therefore, the biosynthesis of coenzyme Q and, particularly, the fungal‐specific protein HorA play a crucial role in virulence of A. fumigatus. Due to its absence in mammals, HorA might represent a novel therapeutic target against fungal infections.