Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co‐occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co‐incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.     

Determinants that govern the recognition and uptake of Escherichia coli O157 : H7 by Acanthamoeba castellanii

Predation by phagocytic predators is a major source of bacterial mortality. The first steps in protozoan predation are recognition and consumption of their bacterial prey. However, the precise mechanisms governing prey recognition and phagocytosis by protists, and the identities of the molecular and cellular factors involved in these processes are, as yet, ill‐characterized. Here, we show that that the ability of the phagocytic bacterivorous amoebae, Acanthamoeba castellanii, to recognize and internalize Escherichia coli, a bacterial prey, varies with LPS structure and composition. The presence of an O‐antigen carbohydrate is not required for uptake of E. coli by A. castellanii. However, O1‐antigen types, not O157 O‐antigen types, inhibit recognition and uptake of bacteria by amoeba. This finding implies that O‐antigen may function as an antipredator defence molecule. Recognition and uptake of E. coli by A. castellanii is mediated by the interaction of mannose‐binding protein located on amoebae's surface with LPS carbohydrate. Phagocytic mammalian cells also use mannose‐binding lectins to recognize and/or mediate phagocytosis of E. coli. Nonetheless, A. castellanii's mannose binding protein apparently displays no sequence similarity with any known metazoan mannose binding protein. Hence, the similarity in bacterial recognition mechanisms of amoebae and mammalian phagocytes may be a result of convergent evolution.     

Free living amoebae could enhance Fusarium oxysporum growth

Free living amoebae and Fusarium oxysporum can be recovered in the same environment and may potentially interact. The presence of these protozoa could lead to an increased development of this filamentous fungus. To assess this potential risk, the interactions between two free living amoebae, Acanthamoeba castellanii and Hartmanella vermiformis, and F. oxysporum, which can be isolated from soil and water, were studied. After 48 hr of coincubation in tap water, culturable fungi were quantified. In addition, the interactions between the free living amoebae and the fungus were investigated using electron microscopy. We show that the presence of amoeba trophozoites increased the growth of F. oxysporum without fungal influence on amoebae viability. In the same way, incubation of the fungus with culture supernatants of the two amoebae induced fungal germination and increased fungal growth. The results of this study confirm that the presence of amoebae should be taken into consideration in the different environments where they may be in contact with Fusarium.     

Differential localization patterns of septins during growth of the human fungal pathogen Aspergillus fumigatus reveal novel functions

Septins, a conserved family of GTPases, are heteropolymeric filament-forming proteins that associate with the cell membrane and cytoskeleton and serve essential functions in cell division and morphogenesis. Their roles in fungal cell wall chitin deposition, septation, cytokinesis, and sporulation have been well established and they have recently been implicated in tissue invasion and virulence in Candida albicans. Septins have never been investigated in the human pathogenic fungus, Aspergillus fumigatus, which is a leading cause of death in immunocompromised patients. Here we localize all the five septins (AspA–E) from A. fumigatus for the first time, and show that each of the five septins exhibit varied patterns of distribution. Interestingly AspE, which is unique to filamentous fungi, and AspD, belonging to the CDC10 class of septins, localized prominently to tubular structures which were dependent on actin and microtubule networks. Localization of AspD and AspE has never been reported in filamentous fungi. Taken together these results suggest that septins in A. fumigatus might have unique functions in morphogenesis and pathogenicity.     

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.     

Aspergillus fumigatus biofilms: Toward understanding how growth as a multicellular network increases antifungal resistance and disease progression

Aspergillus fumigatus is a saprophytic, filamentous fungus found in soils and compost and the causative agent of several pulmonary diseases in humans, birds, and other mammals. A. fumigatus and other filamentous fungi grow as networks of filamentous hyphae that have characteristics of a classic microbial biofilm. These characteristics include production of an extracellular matrix (ECM), surface adhesion, multicellularity, and increased antimicrobial drug resistance. A. fumigatus biofilm growth occurs in vivo at sites of infection, highlighting the importance of defining mechanisms underlying biofilm development and associated emergent properties. We propose that there are 3 distinct phases in the development of A. fumigatus biofilms: biofilm initiation, immature biofilm, and mature biofilm. These stages are defined both temporally and by unique genetic and structural changes over the course of development. Here, we review known mechanisms within each of these stages that contribute to biofilm structure, ECM production, and increased resistance to contemporary antifungal drugs. We highlight gaps in our understanding of biofilm development and function that when addressed are expected to aid in the development of novel antifungal therapies capable of killing filamentous fungal biofilms.     

Melanin targets LC3-associated phagocytosis (LAP): A novel pathogenetic mechanism in fungal disease

Intracellular swelling of conidia of the major human airborne fungal pathogen Aspergillus fumigatus results in surface exposure of immunostimulatory pathogen-associated molecular patterns (PAMPs) and triggers activation of a specialized autophagy pathway called LC3-associated phagocytosis (LAP) to promote fungal killing. We have recently discovered that, apart from PAMPs exposure, cell wall melanin removal during germination of A. fumigatus is a prerequisite for activation of LAP. Importantly, melanin promotes fungal pathogenicity via targeting LAP, as a melanin-deficient A. fumigatus mutant restores its virulence upon conditional inactivation of Atg5 in hematopoietic cells of mice. Mechanistically, fungal cell wall melanin selectively excludes the CYBA/p22phox subunit of NADPH oxidase from the phagosome to inhibit LAP, without interfering with signaling regulating cytokine responses. Notably, inhibition of LAP is a general property of melanin pigments, a finding with broad physiological implications.     

Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum

The aim of this study was to screen certain plant essential oils and active compounds for antifungal activity and their in vitro interaction with fluconazole against drug-resistant pathogenic fungi. The methods employed in this work included disc diffusion, broth macrodilution, time kill methods and checkerboard microtiter tests. Oil compositions were evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. Transmission electron microscopy was used to assess the effect of essential oils on cellular structures of test fungi. Test fungal strains exhibited resistance to at least two drugs (fluconazole and itraconazole). Among the 21 essential oils or active compounds tested, ten showed promising antifungal activity. GC-MS analysis revealed the presence of major active compounds in the essential oils used. Cinnamaldehyde showed the most promising antifungal activity and killing potency against Aspergillus fumigatus MTCC2550 and Trichophyton rubrum IOA-9. Cinnamaldehyde showed strongest synergy with fluconazole against A. fumigatus and T. rubrum by reducing the minimum inhibitory concentration of fluconazole up to 8-fold. Zones of lysis of the cell wall and cell membrane appeared to be where cinnamaldehyde acted on fungi. This study highlights the broad spectrum antifungal activity of essential oils and active compounds and their synergy with fluconazole against drug-resistant fungi.     

Woronin bodies,their impact on stress resistance and virulence of the pathogenic mould Aspergillus fumigatus and their anchoring at the septal pore of filamentous Ascomycota

Hyphae of filamentous Ascomycota consist of compartments that are connected via septal pores. To avoid a dramatic loss of cellular content after wounding, fungi developed mechanisms to occlude their septal pores. In most Pezizomycotina, so‐called Woronin bodies are anchored in proximity to the pore. This is a prominent example for precise spatial positioning of organelles, but so far the underlying molecular organization has remained largely unknown. Using the pathogenic mould Aspergillus fumigatus, we provide evidence that Woronin bodies are important for stress resistance and virulence. Furthermore the molecular machinery anchoring them at the septum is described. Namely, we have identified Lah as the tethering protein and provide evidence that the Woronin body protein HexA binds to the septal pore in a Lah‐dependent manner. Moreover, we demonstrate that a striking poly‐histidine motif targets HexA to the septal cell wall. Thus, the axis HexA‐Lah is an excellent candidate for the tether linking Woronin bodies to the septum. This model applies to A. fumigatus, but most likely also to the vast majority of the Pezizomycotina. Our findings shed light on the evolution of Woronin body anchoring and provide a basis for the development of novel strategies to combat fungal pathogens like A. fumigatus.     

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.     

Feeding Characteristics of an Amoeba (Lobosea: Naegleria) Grazing Upon Cyanobacteria: Food Selection, Ingestion and Digestion Progress

Bacterivory by heterotrophic nanoflagellates and ciliates has been widely studied in aquatic environments, but data on the grazing of amoebae, are still scarce. From the water samples of Dianchi Lake (Kunming, Yunnan Province, China), we isolated an amoeba, designated as Naegleria sp. strain W2, which had potent grazing effects on some kind of cyanobacteria. The food selection mechanism and the digestion process of the amoeba were investigated in batch experiments. Predation experiments showed that filamentous cyanobacteria (e.g., Anabaena, Cylindrospermum, Gloeotrichia, and Phormidium) were readily consumed, with clearance rates ranging from 0.332 to 0.513 nL amoeba⁻¹ h⁻¹. The tight threads (Oscilltoria) and aggregates (Aphanizomenon) could not be ingested; however, their sonicated fragments were observed inside food vacuoles, suggesting that their morphologies prevent them from being ingested. Live video microscopy noted that unicellular Chroococcaceae (e.g., Synechococcus, Aphanocapsa, and Microcystis) were excreted after ingestion, indicating that food selection takes place inside food vacuoles. To determine whether the tastes or the toxins prevented them from being digested, heat-killed cells were retested for predation. Digestion rates and ingestion rates of the amoebae for filamentous cyanobacteria were estimated from food vacuole content volume. Through a “cold-chase” method, we found that the food vacuole contents declined exponentially in diluted amoebae cells, and digestion rates were relatively constant, averaging about 1.5% food vacuole content min⁻¹ at 28°C. Ingestion strongly depended on the satiation status of the amoebae, starved amoebae fed at higher rates compared with satiated amoebae. Our results suggest that the food selection and food processing mechanisms of the amoeba are similar to those of interception feeding flagellates; however, filamentous cyanobacteria cannot obtain a refuge under the grazing pressure of phagotrophic amoebae, which may widen our knowledge on the grazing of protists.     

Fungi,feather damage,and risk of predation

Predation is a powerful selective force with important effects on behavior, morphology, life history, and evolution of prey. Parasites may change body condition, health status, and ability to escape from or defend prey against predators. Once a prey individual has been detected, it can rely on a diversity of means of escape from the pursuit by the predator. Here we tested whether prey of a common raptor differed in terms of fungi from nonprey recorded at the same sites using the goshawk Accipiter gentilis and its avian prey as a model system. We found a positive association between the probability of falling prey to the raptor and the presence and the abundance of fungi. Birds with a specific composition of the community of fungi had higher probability of falling prey to a goshawk than individual hosts with fewer fungi. These findings imply that fungi may play a significant role in predator–prey interactions. The probability of having damaged feathers increased with the number of fungal colonies, and in particular the abundance of Myceliophthora verrucos and Schizophyllum sp. was positively related to the probability of having damaged feathers. In addition, we found a significant correlation between the rate of feather growth of goshawk prey with birds with more fungi being more likely to be depredated. These findings are consistent with the hypothesis that survival and feather quality of birds are related to abundance and diversity of fungi.     

Nondegradative pisatin-resistance inDictyostelium discoideum,Neurospora crassa andNectria haematococca: Similarities and differences

Paradoxically, on pisatin-medium (150 μg/ml) the cellular slime mouldDictyostelium discoideum grows only when plated as spores but not when plated as amoebae. The recent discovery of inducible nondegradativc pisatin resistance in amoebae has allowed us to formulate a model that resolves this paradox. In this model, the germinating amoeba is postulated to acquire a pisatin-resistance phenotype while ensconced within the spore wall. This article reviews the findings on which this model is based and extends it to also account for the differences in pisatin sensitivity phenotype that result from sterol alteration in cellular slime mould^s and fungi.     

Diacylglycerol as a component of the signal-coupling pathway during the initiation of endocytosis in Amoeba proteus

In an attempt to define the transmembrane-signal pathway used to couple external phagocytotic signals with effectors in the cell interior, the effects of diacylglycerol (DG) and related substances were examined in Amoeba proteus. DGs are highly chemotactic, readily attracting amoebae when presented in a glass micropipette. Addition of DG (10^-6 M) to the medium elicits rapid shape changes in the amoeba and the formation of large phagosomes. Monacylglycerol and 1,3-diacylglycerol were much less effective in eliciting phagosome formation. On the assumption that DG was stimulating phosphokinase C (PKC) activity in the amoeba, the effect of phorbol myristate acetate (PMA), a known activator of PKC activity i other cell systems, was assessed in the amoeba. PMA (10^-7 M) alone was capable of bringing about shape changes in amoebae as well as stimulating the formation of phagosomes. These observations suggest that PKC is involved in the signal-coupling associated with the onset of phagocytosis. On the other hand staurosporine and H-7, inhibitors of PKC activity in some cell systems, did not inhibit the phagocytic uptake of Tetrahymena by A. proteus. It may be then that DGs in the amoeba interact directly with elements of the cytoskeleton causing phagosome formation, although a role for PKC in the initiation of phagocytosis in the amoeba cannot be ruled out at this point.     

Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose‐binding proteins

Free‐living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose‐binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose‐binding proteins, Ac–fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.     

A comparative study of endophytic and epiphytic fungal association with leaf of <Emphasis Type="Italic">Eucalyptus citriodora</Emphasis> Hook., and their antimicrobial activity

Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don’t produce sexual spores. The Sorensen’s index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants.     

Description of the marine predator Sericomyxa perlucida gen. et sp. nov., a cultivated representative of the deepest branching lineage of vampyrellid amoebae (Vampyrellida,Rhizaria)

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D’Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage (“clade C”) of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for “clade C.”