Flotillin-Dependent Membrane Microdomains Are Required for Functional Phagolysosomes against Fungal Infections.

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca2+ ions and that inhibition of Ca2+-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.

Mutations in EEA1 are associated with allergic bronchopulmonary aspergillosis and affect phagocytosis of Aspergillus fumigatus by human macrophages.

Allergic bronchopulmonary aspergillosis (ABPA) in asthma is a severe, life-affecting disease that potentially affects over 4.8 million people globally. In the UK, ABPA is predominantly caused by the fungus Aspergillus fumigatus. Phagocytosis is important in clearance of this fungus, and Early Endosome Antigen 1 (EEA1) has been demonstrated to be involved in phagocytosis of fungi. We sought to investigate the role of EEA1 mutations and phagocytosis in ABPA. We used exome sequencing to identify variants in EEA1 associated with ABPA. We then cultured monocyte-derived macrophages (MDMs) from 17 ABPA subjects with A. fumigatus conidia, and analyzed phagocytosis and phagolysosome acidification in relation to the presence of these variants. We found that variants in EEA1 were associated with ABPA and with the rate of phagocytosis of A. fumigatus conidia and the acidification of phagolysosomes. MDMs from ABPA subjects carrying the disease associated genotype showed increased acidification and phagocytosis compared to those from ABPA subjects carrying the non-associated genotypes or healthy controls.The identification of ABPA-associated variants in EEA that have functional effects on MDM phagocytosis and phagolysosome acidification of A. fumigatus conidia revolutionizes our understanding of susceptibility to this disease, which may in future benefit patients by earlier identification or improved treatments. We suggest that the increased phagocytosis and acidification observed demonstrates an over-active MDM profile in these patients, resulting in an exaggerated cellular response to the presence of A. fumigatus in the airways.

The Aspergillus fumigatus conidial melanin production is regulated by the bifunctional bHLH DevR and MADS-box RlmA transcription factors.

Melanins play a crucial role in defending organisms against external stressors. In several pathogenic fungi, including the human pathogen Aspergillus fumigatus, melanin production was shown to contribute to virulence. A. fumigatus produces two different types of melanins, i.e., pyomelanin and dihydroxynaphthalene (DHN)-melanin. DHN-melanin forms the gray-green pigment characteristic for conidia, playing an important role in immune evasion of conidia and thus for fungal virulence. The DHN-melanin biosynthesis pathway is encoded by six genes organized in a cluster with the polyketide synthase gene pksP as a core element. Here, cross-species promoter analysis identified specific DNA binding sites in the DHN-melanin biosynthesis genes pksP-arp1 intergenic region that can be recognized by bHLH and MADS-box transcriptional regulators. Independent deletion of two genes coding for the transcription factors DevR (bHLH) and RlmA (MADS-box) interfered with sporulation and reduced the expression of the DHN-melanin gene cluster. In vitro and in vivo experiments proved that these transcription factors cooperatively regulate pksP expression acting both as repressors and activators in a mutually exclusive manner. The dual role executed by each regulator depends on specific DNA motifs recognized in the pksP promoter region.

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.

Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells.

Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus.

The environmentally acquired fungal pathogen Aspergillus fumigatus causes a variety of severe diseases. Furthermore, it is often found colonizing the respiratory tract of patients suffering from cystic fibrosis. Conidia of this filamentous fungus adhere to substrate surfaces and germinate to form biofilms comprised of dense hyphal networks embedded in an adhesive extracellular matrix (ECM), built predominantly of polysaccharides. These fungal microconsortia are likely to be of clinical relevance, as they have also been observed during growth in the host and they confer drastically reduced susceptibility to antifungals. Little is known about environmental factors or signals contributing to the formation and structural organization of this polysaccharide matrix. Extracellular DNA (eDNA) is an abundant molecule in the mucus-rich surfaces in the lungs of cystic fibrosis patients. Here, we studied its influence on the biofilm establishment and progression of A. fumigatus. Using an in vitro biofilm model eDNA was identified as an efficient biofilm inducer promoting conidial surface adhesion and polysaccharide ECM production. Confocal laser scanning microscopy revealed entirely different ECM architectures depending on the substrates used for biofilm induction. In the presence of serum, adhesive polysaccharides were mainly localized to the hyphal tips appearing as cohesive threads or "halo" areas agglutinating the hyphae. Exogenous DNA altered the structural organization of the biofilm specifically by colocalizing to a grid-like bottom layer of ECM. These results indicate that biofilm formation in A. fumigatus is shaped by certain substrates and in response to host environmental signals.

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.

Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus Aspergillus fumigatus Interferes with the Host Endocytosis Pathway.

Aspergillus fumigatus is the most important air-borne fungal pathogen of humans. The interaction of the pathogen with the host's immune system represents a key process to understand pathogenicity. For elimination of invading microorganisms, they need to be efficiently phagocytosed and located in acidified phagolysosomes. However, as shown previously, A. fumigatus is able to manipulate the formation of functional phagolysosomes. Here, we demonstrate that in contrast to pigmentless pksP mutant conidia of A. fumigatus, the gray-green wild-type conidia inhibit the acidification of phagolysosomes of alveolar macrophages, monocyte-derived macrophages, and human neutrophil granulocytes. Therefore, this inhibition is independent of the cell type and applies to the major immune effector cells required for defense against A. fumigatus. Studies with melanin ghosts indicate that the inhibitory effect of wild-type conidia is due to their dihydroxynaphthalene (DHN)-melanin covering the conidia, whereas the hydrophobin RodA rodlet layer plays no role in this process. This is also supported by the observation that pksP conidia still exhibit the RodA hydrophobin layer, as shown by scanning electron microscopy. Mutants defective in different steps of the DHN-melanin biosynthesis showed stronger inhibition than pksP mutant conidia but lower inhibition than wild-type conidia. Moreover, A. fumigatus and A. flavus led to a stronger inhibition of phagolysosomal acidification than A. nidulans and A. terreus. These data indicate that a certain type of DHN-melanin that is different in the various Aspergillus species, is required for maximal inhibition of phagolysosomal acidification. Finally, we identified the vacuolar ATPase (vATPase) as potential target for A. fumigatus based on the finding that addition of bafilomycin which inhibits vATPase, led to complete inhibition of the acidification whereas the fusion of phagosomes containing wild-type conidia and lysosomes was not affected.

Automated image analysis of the host-pathogen interaction between phagocytes and Aspergillus fumigatus.

Aspergillus fumigatus is a ubiquitous airborne fungus and opportunistic human pathogen. In immunocompromised hosts, the fungus can cause life-threatening diseases like invasive pulmonary aspergillosis. Since the incidence of fungal systemic infections drastically increased over the last years, it is a major goal to investigate the pathobiology of A. fumigatus and in particular the interactions of A. fumigatus conidia with immune cells. Many of these studies include the activity of immune effector cells, in particular of macrophages, when they are confronted with conidia of A. fumigus wild-type and mutant strains. Here, we report the development of an automated analysis of confocal laser scanning microscopy images from macrophages coincubated with different A. fumigatus strains. At present, microscopy images are often analysed manually, including cell counting and determination of interrelations between cells, which is very time consuming and error-prone. Automation of this process overcomes these disadvantages and standardises the analysis, which is a prerequisite for further systems biological studies including mathematical modeling of the infection process. For this purpose, the cells in our experimental setup were differentially stained and monitored by confocal laser scanning microscopy. To perform the image analysis in an automatic fashion, we developed a ruleset that is generally applicable to phagocytosis assays and in the present case was processed by the software Definiens Developer XD. As a result of a complete image analysis we obtained features such as size, shape, number of cells and cell-cell contacts. The analysis reported here, reveals that different mutants of A. fumigatus have a major influence on the ability of macrophages to adhere and to phagocytose the respective conidia. In particular, we observe that the phagocytosis ratio and the aggregation behaviour of pksP mutant compared to wild-type conidia are both significantly increased.

Phagocytosis of melanized Aspergillus conidia by macrophages exerts cytoprotective effects by sustained PI3K/Akt signalling.

Host cell death is a critical component of innate immunity and often determines the progression and outcome of infections. The opportunistic human pathogen Aspergillus fumigatus can manipulate the immune system either by inducing or by inhibiting host cell apoptosis dependent on its distinct morphological form. Here, we show that conidia of Aspergillus ssp. inhibit apoptosis of macrophages induced via the intrinsic (staurosporine) and extrinsic (Fas ligand) pathway. Hence, mitochondrial cytochrome c release and caspase activation were prevented. We further found that the anti-apoptotic effect depends on both host cell de novo protein synthesis and phagocytosis of conidia by macrophages. Moreover, sustained PI3K/Akt signalling in infected cells is an important determinant to resist apoptosis. We demonstrate that pigmentless pksP mutant conidia of A. fumigatus failed to trigger protection against apoptosis and provide evidence that the sustained survival of infected macrophages depends on the presence of the grey-green conidial pigment consisting of dihydroxynaphthalene-melanin. In conclusion, we revealed a novel potential function of melanin in the pathogenesis of A. fumigatus. For the first time, we show that melanin itself is a crucial component to inhibit macrophage apoptosis which may contribute to dissemination of the fungus within the host.

A block of endocytosis of the yeast cell wall integrity sensors Wsc1 and Wsc2 results in reduced fitness in vivo.

The response to cell surface stress in yeast is mediated by a set of five plasma membrane sensors. We here address the relation of intracellular localization of the sensors Wsc1, Wsc2, and Mid2 to their turnover and signaling function. Growth competition experiments indicate that Wsc2 plays an important role in addition to Wsc1 and Mid2. The two Wsc sensors appear at the bud neck during cytokinesis and employ different routes of endocytosis, which govern their turnover. Whereas Wsc1 uses a clathrin-dependent NPFDD signal, Wsc2 relies on a specific lysine residue (K495). In end3 and doa4 endocytosis mutants, both sensors accumulate at the plasma membrane, and a hypersensitivity to cell wall-specific drugs and to treatment with zymolyase is observed. A haploid strain in which endocytosis of the two sensors is specifically blocked displays a reduced fitness in growth competition experiments. If the Mid2 sensor is mobilized by the addition of an endocytosis signal, it mimics the dynamic distribution of the Wsc sensors, but is unable to complement the specific growth defects of a wsc1 deletion. These data suggest that sensor distribution is not the major determinant for its specificity.

Interaction of phagocytes with filamentous fungi.

Phagocytosis of conidia by macrophages and destruction of hyphae by neutrophils are key processes in the defense against infections caused by filamentous fungi. Impairment in phagocytic function leads to increased susceptibility for an infection with Aspergillus species. The fact that a Th1-based immune response to an infection with Aspergillus species results in an improved prognosis for survival underlines the importance of the phagocytic response. Recognition of conidia by macrophages occurs after shedding of the hydrophobic rodlet layer during swelling and germination. Whereas Aspergillus conidia are killed by various immune effector cells, hyphae are in particular targeted and killed by neutrophils. Moreover, both conidia and hyphae are trapped in neutrophil extracellular traps (NETs) that form a containment to localize the infection and to prevent systemic spreading of the fungus in the host. In addition, A. fumigatus interferes with the innate immunity, with both the complement system and defense mechanisms of phagocytes, thereby evading at least in part the innate immune system.

Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.