Redundant synthesis of a conidial polyketide by two distinct secondary metabolite clusters in Aspergillus fumigatus

Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non‐reducing polyketide synthase‐encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10‐fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non‐reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes.     

Indoleamine 2,3-Dioxygenase Is Involved in the Inflammation Response of Corneal Epithelial Cells to Aspergillus fumigatus Infections

Indoleamine 2,3-dioxygenase (IDO), which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs), known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with Aspergillus fumigatus (A. fumigatus) and HCECs incubated with spores of A. fumigatus. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT) enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by A. fumigatus spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with A. fumigatus stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by A. fumigatus. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to A. fumigatus spores-induced up-regulation of IDO.     

Fungal Secondary Metabolites and Their Fundamental Roles in Human Mycoses

Understanding which fungal factors allow colonization and infection of a human host is critical to lowering the incidence of human mycoses and related mortalities. In the pathogen Aspergillus fumigatus, secondary metabolites, small bioactive molecules produced by many opportunistic fungal pathogens, have important roles in suppressing and providing protection from host defenses. Deletion of LaeA, a global regulator of secondary metabolism in fungi, significantly decreases A. fumigatus virulence, in part owing to loss of gliotoxin and hydrophobin production. In addition to gliotoxin, dihydroxynaphthalene (DHN) melanin and siderophores are other A. fumigatus virulence factors; all three metabolites are derived from hallmark secondary metabolite gene clusters. Many of the gene clusters producing toxin metabolites have yet to be deciphered, and the study of secondary metabolites and their role in the virulence of human pathogens is a nascent field.     

High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence

Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo‐dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and β‐1,3‐glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus.     

The ZrfC alkaline zinc transporter is required for Aspergillus fumigatus virulence and its growth in the presence of the Zn/Mn‐chelating protein calprotectin

Aspergillus fumigatus can invade the lungs of immunocompromised individuals causing a life‐threatening disease called invasive pulmonary aspergillosis (IPA). To grow in the lungs, A. fumigatus obtains from the host all nutrients, including zinc. In living tissues, however, most zinc is tightly bound to zinc‐binding proteins. Moreover, during infection the bioavailability of zinc can be further decreased by calprotectin, an antimicrobial Zn/Mn‐chelating protein that is released by neutrophils in abscesses. Nevertheless, A. fumigatus manages to uptake zinc from and grow within the lungs of susceptible individuals. Thus, in this study we investigated the role of the zrfA, zrfB and zrfC genes, encoding plasma membrane zinc transporters, in A. fumigatus virulence. We showed that zrfC is essential for virulence in the absence of zrfA and zrfB, which contribute to fungal pathogenesis to a lesser extent than zrfC and are dispensable for virulence in the presence of zrfC. The special ability of ZrfC to scavenge and uptake zinc efficiently from lungtissue depended on its N‐terminus, which is absent in the ZrfA and ZrfB transporters. In addition, under Zn‐ and/or Mn‐limiting conditions zrfC enables A. fumigatus to grow in the presence of calprotectin, which is detected in fungal abscesses of non‐leucopenic animals. This study extends our knowledge about the pathobiology of A. fumigatus and suggests that fungal zinc uptake could be a promising target for new antifungals.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.     

Recurrent Aspergillus contamination in a biomedical research facility: a case study

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.     

Quantification of airborne Aspergillus fumigatus spores in rabbit houses by real-time polymerase chain reaction

To accurately quantify airborne Aspergillus fumigatus (A. fumigatus) spores in rabbit houses, the real-time polymerase chain reaction (real-time PCR) and culture-based counting method (CCM) were employed to determine the airborne A. fumigatus spore concentrations. The results showed that, of the three rabbit houses (A, B, and C), the average concentrations of airborne A. fumigatus spores determined by real-time PCR were 3.0 × 10³, 3.3 × 10³, and 1.5 × 10³ spores/m³ air, respectively, while those determined by CCM were 2.5 × 10², 2.8 × 10², and 1.1 × 10² colony-forming unit/m³ air (CFU/m³ air), respectively, i.e., the former concentration was 12–14 times higher than the latter one. Therefore, the conventional CCM underestimated the concentrations of airborne fungal spores, and it is insufficient to determine the microbial aerosol concentration and evaluate the health risk only using CCM.     

The different morphologies of yeast and filamentous fungi trigger distinct killing and feeding mechanisms in a fungivorous amoeba

Size and diverse morphologies pose a primary challenge for phagocytes such as innate immune cells and predatory amoebae when encountering fungal prey. Although filamentous fungi can escape phagocytic killing by pure physical constraints, unicellular spores and yeasts can mask molecular surface patterns or arrest phagocytic processing. Here, we show that the fungivorous amoeba Protostelium aurantium was able to adjust its killing and feeding mechanisms to these different cell shapes. Yeast-like fungi from the major fungal groups of basidiomycetes and ascomycetes were readily internalized by phagocytosis, except for the human pathogen Candida albicans whose mannoprotein coat was essential to escape recognition by the amoeba. Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized, but swelling and the onset of germination induced internalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip and killed by an actin-mediated invasion of fungal filaments. Our results demonstrate that predatory pressure imposed by amoebae in natural environments selects for distinct survival strategies in yeast and filamentous fungi but commonly targets the fungal cell wall as a crucial molecular pattern associated to prey and pathogens.     

The variation in spore concentrations of selected fungal taxa associated with weather conditions in Szczecin,Poland, 2004–2006

The investigation of airborne fungal spore concentrations was carried out in Szczecin, Poland between 2004 and 2006. The objective of the studies was to determine a seasonal variation in concentrations of selected fungal spore types due to meteorological parameters. The presence of spores of ten taxa: Cladosporium, Ganoderma, Alternaria, Epicoccum, Didymella, Torula, Dreschlera‐type, Polythrincium, Stemphylium and Pithomyces was recorded in Szczecin using a volumetric method (Hirst type). Fungal spores were present in the air in large numbers in summer. The highest concentrations were noted in June, July and August. The peak period was recorded in August for most of the studied spore types: Ganoderma, Alternaria, Epicoccum, Dreschlera‐type, Polythrincium and Stemphylium. Cladosporium and Didymella spores reached their highest concentrations in July while concentrations of Torula were highest in May and Pithomyces in September. Multiple regression analysis was performed for three fungal seasons: 2004, 2005, and 2006. Spore concentrations were positively correlated with minimum temperature for seven spore types in 2004, for five spore types in 2005, and for eight spore types in 2006 (significance level of α = 0.05). Some spore types are also significantly correlation among their concentrations, pressure, relative humidity and rain. Minimum temperature appeared to be the most influential factor for most spore types.     

ULTRASTRUCTURE OF DISPERSED AND IN SITU SPECIMENS OF THE DEVONIAN SPORE RHABDOSPORITES LANGII: EVIDENCE FOR THE EVOLUTIONARY RELATIONSHIPS OF PROGYMNOSPERMS

Abstract: The spore Rhabdosporites (Triletes) langii (Eisenack) Richardson, 1960 is abundant and well preserved in Middle Devonian (Eifelian) ‘Middle Old Red Sandstone’ deposits from the Orcadian Basin, Scotland. Here it occurs as dispersed individual spores and in situ in isolated sporangia. This paper reports on a detailed light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of both dispersed and in situ spores. The dispersed spores are pseudosaccate with a thick walled inner body enclosed within an outer layer that was originally attached only over the proximal face. The inner body has lamellate/laminate ultrastructure consisting of fine lamellae that are continuous around the spore and parallel stacked. Towards the outer part of the inner body these group to form thicker laminate structures that are also continuous and parallel stacked. The outer layer has spongy ultrastructure. In situ spores preserved in the isolated sporangia are identical to the dispersed forms in terms of morphology, gross structure and wall ultrastructure. The sporangium wall is two‐layered. A thick coalified outer layer is cellular and represents the main sporangium wall. This layer is readily lost if oxidation is applied during processing. A thin inner layer is interpreted as a peritapetal membrane. This layer survives oxidation as a tightly adherent membranous covering of the spore mass. Ultrastructurally it consists of three layers, with the innermost layer composed of material similar to that comprising the outer layer of the spores. Based on the new LM, SEM and TEM information, consideration is given to spore wall formation. The inner body of the spores is interpreted as developing by centripetal accumulation of lamellae at the plasma membrane. The outer layer is interpreted as forming by accretion of sporopollenin units derived from a tapetum. The inner layer of the sporangium wall is considered to represent a peritapetal membrane formed from the remnants of this tapetum. The spore R. langii derives from aneurophytalean progymnosperms. In light of the new evidence on spore/sporangium characters, and hypotheses of spore wall development based on interpretation of these, the evolutionary relationships of the progymnosperms are considered in terms of their origins and relationship to the seed plants. It is concluded that there is a smooth evolutionary transition between Apiculiretusispora‐type spores of certain basal euphyllophytes, Rhabdosporites‐type spores of aneurophytalean progymnosperms and Geminospora‐/Contagisporites‐type spores of heterosporous archaeopteridalean progymnosperms. Prepollen of basal seed plants (hydrasperman, medullosan and callistophytalean pteridosperms) are easily derived from the spores of either homosporous or heterosporous progymnosperms. The proposed evolutionary transition was sequential with increasing complexity of the spore/pollen wall probably reflecting increasing sophistication of reproductive strategy. The pollen wall of crown group seed plants appears to incorporate a completely new developmental mechanism: tectum and infratectum initiation within a glycocalyx‐like Microspore Surface Coat. It is unclear when this feature evolved, but it appears likely that it was not present in the most basal stem group seed plants.     

Main airborne Ascomycota spores: characterization by culture, spore morphology, ribosomal DNA sequences and enzymatic analysis

The aim of this work was to identify the main allergy-related Ascomycetes fungal spores present in the atmosphere of Porto, using different and complementary techniques. The atmospheric sampling, performed in the atmosphere of Porto (Portugal) from August 2006 to July 2008, indicated Cladosporium, Penicillium, Aspergillus and Alternaria as the main fungal spore taxa. Alternaria and Cladosporium peaks were registered during summer. Aspergillus and Penicillium highest values were registered from late winter to early spring. Additionally, the Andersen sampler allowed the culture and isolation of the collected viable spores subsequently used for different identification approaches. The internal-transcribed spacer region of the nuclear ribosomal repeat unit sequences of airborne Ascomycetes fungi isolates revealed 11 taxonomically related fungal species. Among the identified taxa, Penicillum and Aspergillus presented the highest diversity, while only one species of Cladosporium and Alternaria, respectively, were identified. All selected fungal spore taxa possessed phosphatase, esterase, leucine arylamidase and β-glucosidase enzymatic activity, while none had lipase, cystine arylamidase, trypsin or β-glucuronidase activity. The association between the spore cell wall morphology, DNA-based techniques and enzymatic activity approaches allowed a more reliable identification procedure of the airborne Ascomycota fungal spores.     

Effects of weather conditions on selected airborne fungal spores in the southern part of the state of Enugu,Nigeria

A study of airborne fungal spore was carried out at nine locations in the southern part of the state of Enugu, Nigeria, from March 2005 to February 2006. The aim of the study was to ascertain the variations in selected fungal spore types at the sites owing to weather conditions. The variation in airborne fungal spores of 14 taxa was studied using modified Tauber pollen traps including Alternaria, Corynespora, Curvularia, Drechslera type, Endophragmiella, Botryodiplodia, Ganoderma, Gliomastrix, Nigrospora, Pithomyces, Spegazzinia, Sporidesmium, Tetraploa and Ustilago. The frequency of the spore types recorded showed considerable variation. The highest spore counts were recorded in July, June and October. The highest numbers of fungal spores were recorded during the rainy season (June–October) to early dry season (November–December). The peak of occurrence of most selected fungal spore types was July. The highest percentages of fungal spores were documented at the recording stations Mgbowo Junction, UNTH Ituku Ozalla and Oji River Express Junction. Spearman’s correlation analyses were performed for the monthly amounts of the fungal spore types and monthly meteorological factors. The numbers of Curvularia, Nigrospora and Sporidesmium was significantly correlated with relative humidity, while those of Endophragmiella, Pithomyces and Nigrospora were significantly correlated with temperature. A significant correlation was also found between the number of Nigrospora spores and light intensity and Sporidesmium spores and wind velocity. Relative humidity and temperature seem to be the most important weather conditions affecting the frequency of the selected spore types in the atmosphere.     

Pilot production of Clonostachys rosea conidia in a solid‐state fermentor optimized using response surface methodology

The nonpathogenic, saprophytic fungus Clonostachys rosea is one of the most powerful fungal biological control agents (BCAs). However, the production of fungal BCAs is still a major constraint for their large‐scale use and commercialization. Here, we developed a novel solid‐fermentation reactor that is light transparent and ventilated both at the top and the bottom, and optimized C. rosea cultivation conditions in solid‐state fermentation using response surface methodology. The growth area of spores provided by the novel fermentor was two times that of the traditional one. A quadratic polynomial model was developed, which indicated the effects of variables on the conidia yield. The greatest spore production of 3.50 × 10¹⁰ spores/g‐dry‐matter was obtained after 11 days at the initial moisture content of 69.2% w/w, the medium thickness of 3.84 cm, and the porosity of 0.37%. The optimized spore yield was increased by one order of magnitude. The fermentation time was shortened from 15 to 11 days. With the novel solid‐fermentation reactor, increase in C. rosea spores production and decrease in fermentation time were achieved. Current data imply that both the novel solid‐fermentation reactor designed and the optimized fermentation conditions are suitable for industrial‐scale C. rosea spore production.     

Low-Volume Toolbox for the Discovery of Immunosuppressive Fungal Secondary Metabolites

The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.     

Fungal spore swelling and germination are restricted by the macrophage phagolysosome

Many species of medically important fungi are prolific in the formation of asexual spores. Spores undergo a process of active swelling and cell wall remodelling before a germ tube is formed and filamentous growth ensues. Highly elongated germ tubes are known to be difficult to phagocytose and pose particular challenges for immune phagocytes. However, the significance of the earliest stages of spore germination during immune cell interactions has not been investigated and yet this is likely to be important for defence against sporogenous fungal pathogens. We show here that macrophages restrict the early phases of the spore germination process of Aspergillus fumigatus and Mucor circinelloides including the initial phase of spore swelling, spore germination and early polarised growth. Macrophages are therefore adept at retarding germination as well as subsequent vegetative growth which is likely to be critical for immune surveillance and protection against sporulating fungi.     

MonitoringAspergillus fumigatus aerosols from a composting facility

The large, outdoor Islip Yard Waste Composting Facility on Long Island, New York was investigated as a source of airborne fungus spores. The Burkard-Hirst volumetric spore trap was used for the first extensive sampling of small mold spores for this application. Samplers were operated continuously from 21 August to 30 November 1992 in the facility and in a suburban community about 540 m from the facility. A control site approximately 10 000 m from the facility was also sampled to establish background levels of fungus spores. The facility site had higher average readings ofAspergillus fumigatus spores than did the community and both were higher than the control.A. fumigatus was the only fungus among 30 categories tracked that differed significantly between the facility and control sites. It was also isolated repeatedly from the compost. Higher average levels ofA. fumigatus were measured in the community when winds blew from the facility, and also during times when the compost was moved or mixed at the facility. No correlation was found between wind direction or work times andA. fumigatus conidia at the control site. The study shows that this compost facility can produce a measurable increase in the number of airborneA. fumigatus conidia both at the edge of the facility and at 540 m downwind. It also demonstrates that the Burkard spore trap can be used for monitoring small, airborne mold spores, but it is a difficult and labor intensive task.