Aspergillus fumigatus MedA governs adherence,host cell interactions and virulence

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusΔmedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The ΔmedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro‐inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusΔmedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.     

The signal peptide peptidase SppA is involved in sterol regulatory element‐binding protein cleavage and hypoxia adaptation in Aspergillus nidulans

Using forward genetics, we revealed that the signal peptide peptidase (SPP) SppA, an aspartyl protease involved in regulated intramembrane proteolysis (RIP), is essential for hypoxia adaptation in Aspergillus nidulans, as well as hypoxia‐sensitive mutant alleles of a sterol regulatory element‐binding protein (SREBP) srbA and the Dsc ubiquitin E3 ligase complex dscA‐E. Both null and dead activity [D337A] mutants of sppA failed to grow in hypoxia, and the growth defect of ΔsppA was complemented by nuclear SrbA‐N381 expression. Additionally, SppA interacted with SrbA in the endoplasmic reticulum, where SppA localized in normoxia and hypoxia. Expression of the truncated SrbA‐N414 covering the SrbA sequence prior to the second transmembrane region rescued the growth of ΔdscA but not of ΔsppA in hypoxia. Unlike ΔdscA and ΔdscA;ΔsppA double mutants, in which SrbA cleavage was blocked, the molecular weight of cleaved SrbA increased in ΔsppA compared to the control strain in immunoblot analyses. Overall, our data demonstrate the sequential cleavage of SrbA by Dsc‐linked proteolysis followed by SppA, proposing a new model of RIP for SREBP cleavage in fungal hypoxia adaptation. Furthermore, the function of SppA in hypoxia adaptation was consistent in Aspergillus fumigatus, suggesting the potential roles of SppA in fungal pathogenesis.     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth,morphogenesis and viability

O‐mannosylation is an essential protein modification in eukaryotes. It is initiated at the endoplasmic reticulum by O‐mannosyltransferases (PMT) that are evolutionary conserved from yeast to humans. The PMT family is phylogenetically classified into PMT1, PMT2 and PMT4 subfamilies, which differ in protein substrate specificity and number of genes per subfamily. In this study, we characterized for the first time the whole PMT family of a pathogenic filamentous fungus, Aspergillus fumigatus. Genome analysis showed that only one member of each subfamily is present in A. fumigatus, PMT1, PMT2 and PMT4. Despite the fact that all PMTs are transmembrane proteins with conserved peptide motifs, the phenotype of each PMT deletion mutant was very different in A. fumigatus. If disruption of PMT1 did not reveal any phenotype, deletion of PMT2 was lethal. Disruption of PMT4 resulted in abnormal mycelial growth and highly reduced conidiation associated to significant proteomic changes. The double pmt1pmt4 mutant was lethal. The single pmt4 mutant exhibited an exquisite sensitivity to echinocandins that is associated to major changes in the expression of signal transduction cascade genes. These results indicate that the PMT family members play a major role in growth, morphogenesis and viability of A. fumigatus.     

Cloning,expression and purification of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

Abstract

The antimalarial drugs are of fundamental importance in the control of malaria, especially for the lack of efficient treatments and acquired resistance to the existing drugs. For this reason, there is a continuous work in identifying novel, less toxic and effective chemotherapies as well as new therapeutic targets against the causative agents of malaria. In this context, a superfamily of metalloenzymes named carbonic anhydrases (CAs, EC 4.2.1.1) has aroused a great interest as druggable enzymes to limit the development of Plasmodium falciparum gametocytes. CAs catalyze a common reaction in all life domains, the carbon dioxide hydration to bicarbonate and protons (CO₂?+?H₂O ? HCO₃^-?+?H⁺). P. falciparum synthesizes pyrimidines de novo starting from HCO₃^-, which is generated from CO₂ through the action of the η-CA identified in the genome of the protozoan. Here, we propose a procedure for the preparation of a wider portion of the protozoan η-CA, named PfCAdom (358 amino acid residues), with respect to the truncated form prepared by Krungkrai et al. (PfCA1, 235 amino acid residues). The results evidenced that the recombinant PfCAdom, produced as a His-tag fusion protein, was 2.7 times more active with respect the truncated form PfCA1.     

Functional analysis of the superoxide dismutase family in Aspergillus fumigatus

Reactive oxidant species produced by phagocytes have been reported as being involved in the killing of Aspergillus fumigatus. Fungal superoxide dismutases (SODs) that detoxify superoxide anions could be putative virulence factors for this opportunistic pathogen. Four genes encoding putative Sods have been identified in the A. fumigatus genome: a cytoplasmic Cu/ZnSOD (AfSod1p), a mitochondrial MnSOD (AfSod2p), a cytoplasmic MnSOD (AfSod3p) and AfSod4 displaying a MnSOD C‐terminal domain. During growth, AfSOD1 and AfSOD2 were highly expressed in conidia whereas AfSOD3 was only strongly expressed in mycelium. AfSOD4 was weakly expressed compared with other SODs. The deletion of AfSOD4 was lethal. Δsod1 and Δsod2 mutants showed a growth inhibition at high temperature and a hypersensitivity to menadione whereas the sod3 mutant had only a slight growth delay at high temperature. Multiple mutations had only an additive effect on the phenotype. The triple sod1/sod2/sod3 mutant was characterized by a delay in conidial germination, a reduced conidial survival during storage overtime, the highest sensitivity to menadione and an increased sensitivity to killing by alveolar macrophage of immunocompetent mice. In spite of these phenotypes, no significant virulence difference was observed between the triple mutant and parental strain in experimental murine aspergillosis models in immunocompromised animals.     

Fruit,flies and filamentous fungi – experimental analysis of animal–microbe competition using Drosophila melanogaster and Aspergillus mould as a model system

In addition to their fundamental role in nutrient recycling, saprobiotic microorganisms may be considered as typical consumers of food‐limited ephemeral resource patches. As such, they may be engaged in inter‐specific competition with saprophagous animals feeding from the same resource. Bacteria and filamentous fungi are known to synthesise secondary metabolites, some of which are toxic and have been proposed to deter or harm animals. The microorganisms may, however, also be negatively affected if saprophagous animals do not avoid microbe‐laden resources but feed in the presence of microbial competitors. We hypothesised that filamentous fungi compete with saprophagous insects, whereby secondary metabolites provide a chemical shield against the insect competitors. For testing this, we developed a new ecological model system representing a case of animal–microbe competition between saprobiotic organisms, comprising Drosophila melanogaster and species of the fungus Aspergillus (A. nidulans, A. fumigatus, A. flavus). Infestation of Drosophila breeding substrate with proliferating fungal colonies caused graduated larval mortality that strongly depended on mould species and colony age. Confrontation with conidiospores only, did not result in significant changes in larval survival, suggesting that insect death may not be ascribed to pathogenic effects. When confronted with colonies of transgenic fungi that lack the ability to express the global secondary metabolite regulator LaeA (ΔlaeA), larval mortality was significantly reduced compared to the impact of the wild type strains. Yet, also in the ΔlaeA strains, inter‐specific variation in the influence on insect growth occurred. Competition with Drosophila larvae impaired fungal growth, however, wild type colonies of A. nidulans and A. flavus recovered more rapidly from insect competition than the corresponding ΔlaeA mutants (not in A. fumigatus). Our findings provide genetic evidence that toxic secondary metabolites synthesised by saprotrophic fungi may serve as a means to combat insect competitors. Variation in the ability of LaeA to control expression of various secondary metabolite gene clusters might explain the observed species‐specific variation in Drosophila–Aspergillus competition.     

Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus

The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S‐transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA‐deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H₂O₂, diamide, and 4,4′‐dipyridyl disulfide (DPS) than the wild‐type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol‐3‐phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild‐type. The differential expression of two aminoacyl‐tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin‐bundling protein Sac6 and vacuolar dynamin‐like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed.     

Utilization of inorganic carbon in the edible cyanobacterium Ge-Xian-Mi (Nostoc) and its role in alleviating photo-inhibition

The present work investigated the inorganic carbon (C_i) uptake, fluorescence quenching and photo‐inhibition of the edible cyanobacterium Ge‐Xian‐Mi (Nostoc) to obtain an insight into the role of CO₂ concentrating mechanism (CCM) operation in alleviating photo‐inhibition. Ge‐Xian‐Mi used HCO₃^– in addition to CO₂ for its photosynthesis and oxygen evolution was greater than the theoretical rates of CO₂ production derived from uncatalysed dehydration of HCO₃^–. Multiple transporters for CO₂ and HCO₃^– operated in air‐grown Ge‐Xian‐Mi. Na⁺‐dependent HCO₃^– transport was the primary mode of active C_i uptake and contributed 53–62% of net photosynthetic activity at 250 µmol L^?1 KHCO₃ and pH 8.0. However, the CO₂‐uptake systems and Na⁺‐independent HCO₃^– transport played minor roles in Ge‐Xian‐Mi and supported, respectively, 39 and 8% of net photosynthetic activity. The steady‐state fluorescence decreased and the photochemical quenching increased in response to the transport‐mediated accumulation of intracellular C_i. Inorganic carbon transport was a major factor in facilitating quenching during the initial stage and the initial rate of fluorescence quenching in the presence of iodoacetamide, an inhibitor of CO₂ fixation, was 88% of control. Both the initial rate and extent of fluorescence quenching increased with increasing external dissolved inorganic carbon (DIC) and saturated at higher than 200 µmol L^?1 HCO₃^–. The operation of the CCM in Ge‐Xian‐Mi served as a means of diminishing photodynamic damage by dissipating excess light energy and higher external DIC in the range of 100–10000 µmol L^?1 KHCO₃ was associated with more severe photo‐inhibition under strong irradiance.     

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.     

The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus

As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen-activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Δmsb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Δmsb2^C) in A. flavus. Using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down-regulated aflatoxin B₁ (AFB₁) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Δmsb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Δmsb2^C. Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.     

BsdABsd2‐dependent vacuolar turnover of a misfolded version of the UapA transporter along the secretory pathway: prominent role of selective autophagy

Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non‐endocytic vacuolar turnover of ΔR481 is dependent on BsdA^Bsd2, an ER transmembrane adaptor of HulA^Rsp5 ubiquitin ligase. We obtain in vivo evidence that BsdA^Bsd2 interacts with HulA^Rsp5 and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO^ts) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes.     

The role of extracellular carbonic anhydrase for accumulation of inorganic carbon in the green alga Chlamydomonas reinhardtii. A comparison between wild-type and cell-wall-less mutant cells

The role of extracellular carbonic anhydrase (CA_ex) for dissolved inorganic carbon (DIC) accumulation in the green alga Chlamydomonas reinhardtii was investigated. It was found that when algal cells were bubbled with ambient air, cell-wall-less mutant cells exhibited the same high photosynthetic affinity for CO₂ as wild-type cells despite a 10 times lower activity of CA^ex. It was also found that the affinity for CO₂ was further increased when the total DIC concentration of the algal medium was reduced from that in equilibrium with ambient air to even lower levels. This increased affinity was not correlated with any further increase in the CA_ex activity. Dextran-bound sulfonamide (DBS. 100 μM bound ligand) completely inhibited the activity of CA_ex in intact, low-DIC grown, wild-type cells, while photosynthesis at <2 μM CO₂(aq) proceeded at a far greater rate than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO^?₃. DBS-inhibition of CA_ex, during the induction of the DIC-accumulating mechanism in previously high-DIC grown cells, only caused a 50% inhibition of photosynthesis at 10 μM CO₂(aq) after 1 h of low-DIC acclimation. It was also shown that 50 μM acetazolamide (AZ) inhibited photosynthesis at low DIC concentrations to a relatively higher degree than DBS, suggesting that AZ inhibited intracellular CA as well. Taken together, these results suggest that low-DIC grown cells of C. reinhardtii have the ability to transport HCO^?₃ across the plasma membrane in addition to the CA_ex-mediated, facilitated diffusion and/or transport of CO₂. It is also suggested that the relative importance of these two fluxes (CO₂ or HCO^?₃) is dependent on the growth and experimental conditions. Facilitated CO₂ uptake seems to be most prevalent, supported by HCO^?₃-transport under more or less extreme situations, such as a reduction of CO₂ to extremely low concentrations, leakage of CA_ex to the medium as in cultures of cell-wall-less mutant cells or when the activity of CA_ex has been artificially inhibited.