Macrophages inhibit extracellular hyphal growth of A. fumigatus through Rac2 GTPase signaling.

Macrophages act as a first line of defense against pathogens. Against Aspergillus fumigatus, a fungus with pathogenic potential in immunocompromised patients, macrophages can phagocytose fungal spores and inhibit spore germination to prevent the development of tissue-invasive hyphae. However, the cellular pathways that macrophages use to accomplish these tasks and any roles macrophages have later in infection against invasive forms of fungi are still not fully known. Rac-family Rho GTPases are signaling hubs for multiple cellular functions in leukocytes, including cell migration, phagocytosis, reactive oxygen species (ROS) generation, and transcriptional activation. We therefore aimed to further characterize the function of macrophages against A. fumigatus in an in vivo vertebrate infection model by live imaging of the macrophage behavior in A. fumigatus-infected rac2 mutant zebrafish larvae. While Rac2-deficient zebrafish larvae are susceptible to A. fumigatus infection, Rac2 deficiency does not impair macrophage migration to the infection site, interaction with and phagocytosis of spores, spore trafficking to acidified compartments, or spore killing. However, we reveal a role for Rac2 in macrophage-mediated inhibition of spore germination and control of invasive hyphae. Re-expression of Rac2 under a macrophage-specific promoter rescues the survival of A. fumigatus-infected rac2 mutant larvae through increased control of germination and hyphal growth. Altogether, we describe a new role for macrophages against extracellular hyphal growth of A. fumigatus and report that the function of the Rac2 Rho GTPase in macrophages is required for this function.

Oxymatrine mitigates Aspergillus fumigatus keratitis by suppressing fungal activity and restricting pyroptosis.

Fungal keratitis (FK) is a refractory keratitis caused by excessive inflammation and fungal damage. Excessive inflammation can lead to tissue damage and corneal opacity, resulting in a poor prognosis for FK. Oxymatrine (OMT) is a natural alkaloid, which has rich pharmacological effects, such as antioxidant and anti-inflammation. However, its antifungal activity and the mechanism of action in FK have not been elucidated. This study confirmed that OMT suppressed Aspergillus fumigatus growth, biofilm formation, the integrity of fungal cell and conidial adherence. OMT not only effectively reduced corneal fungal load but also inflammation responses. OMT lessened the recruitment of neutrophils and macrophages in FK. In addition, OMT up-regulated the expression of Nrf2 and down-regulated the expression of IL-18, IL-1ß, caspase-1, NLRP3 and GSDMD. Pre-treatment with Nrf2 inhibitor up-regulated the expression of IL-1ß, IL-18, caspase-1, NLRP3 and GSDMD supressed by OMT. In conclusion, OMT has efficient anti-inflammatory and antifungal effects by suppressing fungal activity and restricting pyroptosis via Nrf2 pathway. OMT is considered as a potential option for the treatment of FK.

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.

NADPH Oxidase Limits Collaborative Pattern-Recognition Receptor Signaling to Regulate Neutrophil Cytokine Production in Response to Fungal Pathogen-Associated Molecular Patterns.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by genetic defects in leukocyte NADPH oxidase, which has both microbicidal and immunomodulatory roles. Hence, CGD is characterized by recurrent bacterial and fungal infections as well as aberrant inflammation. Fungal cell walls induce neutrophilic inflammation in CGD; yet, underlying mechanisms are incompletely understood. This study investigated the receptors and signaling pathways driving aberrant proinflammatory cytokine production in CGD neutrophils activated by fungal cell walls. Although cytokine responses to ß-glucan particles were similar in NADPH oxidase-competent and NADPH oxidase-deficient mouse and human neutrophils, stimulation with zymosan, a more complex fungal particle, induced elevated cytokine production in NADPH oxidase-deficient neutrophils. The dectin-1 C-type lectin receptor, which recognizes ß-glucans (1-3), and TLRs mediated cytokine responses by wild-type murine neutrophils. In the absence of NADPH oxidase, fungal pathogen-associated molecular patterns engaged additional collaborative signaling with Mac-1 and TLRs to markedly increase cytokine production. Mechanistically, this cytokine overproduction is mediated by enhanced proximal activation of tyrosine phosphatase SHP2-Syk and downstream Card9-dependent NF-κB and Card9-independent JNK-c-Jun. This activation and amplified cytokine production were significantly decreased by exogenous H2O2 treatment, enzymatic generation of exogenous H2O2, or Mac-1 blockade. Similar to zymosan, Aspergillus fumigatus conidia induced increased signaling in CGD mouse neutrophils for activation of proinflammatory cytokine production, which also used Mac-1 and was Card9 dependent. This study, to our knowledge, provides new insights into how NADPH oxidase deficiency deregulates neutrophil cytokine production in response to fungal cell walls.

Fungal biofilm architecture produces hypoxic microenvironments that drive antifungal resistance.

Human fungal infections may fail to respond to contemporary antifungal therapies in vivo despite in vitro fungal isolate drug susceptibility. Such a discrepancy between in vitro antimicrobial susceptibility and in vivo treatment outcomes is partially explained by microbes adopting a drug-resistant biofilm mode of growth during infection. The filamentous fungal pathogen Aspergillus fumigatus forms biofilms in vivo, and during biofilm growth it has reduced susceptibility to all three classes of contemporary antifungal drugs. Specific features of filamentous fungal biofilms that drive antifungal drug resistance remain largely unknown. In this study, we applied a fluorescence microscopy approach coupled with transcriptional bioreporters to define spatial and temporal oxygen gradients and single-cell metabolic activity within A. fumigatus biofilms. Oxygen gradients inevitably arise during A. fumigatus biofilm maturation and are both critical for, and the result of, A. fumigatus late-stage biofilm architecture. We observe that these self-induced hypoxic microenvironments not only contribute to filamentous fungal biofilm maturation but also drive resistance to antifungal treatment. Decreasing oxygen levels toward the base of A. fumigatus biofilms increases antifungal drug resistance. Our results define a previously unknown mechanistic link between filamentous fungal biofilm physiology and contemporary antifungal drug resistance. Moreover, we demonstrate that drug resistance mediated by dynamic oxygen gradients, found in many bacterial biofilms, also extends to the fungal kingdom. The conservation of hypoxic drug-resistant niches in bacterial and fungal biofilms is thus a promising target for improving antimicrobial therapy efficacy.

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatus infection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian ß-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.

Kaempferol ameliorate the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting the Dectin-1 and p38 MAPK pathway.

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1ß, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway.

The role of Glabridin in antifungal and anti-inflammation effects in Aspergillus fumigatus keratitis.

PURPOSE: To investigate the effect of Glabridin (GLD) in Aspergillus fumigatus keratitis and its associated mechanisms. METHODS: Aspergillus fumigatus (A. fumigatus) conidia was inoculated in 96-well plate, and minimal inhibitory concentration (MIC) and biofilm formation ability were evaluated after GLD treatment. Spore adhesion ability was evaluated in conidia infected human corneal epithelial cells (HCECs). Keratitis mouse model was created by corneal intrastromal injection with A. fumigatus conidia, and GLD treatment started at the day after infection. The number of fungal colonies was calculated by plate count, and degree of corneal inflammation was assessed by clinical score. Flow cytometry, myeloperoxidase (MPO), and immunofluorescence staining (IFS) experiments were used to assess neutrophil infiltrations. PCR, ELISA and Western blot were conducted to determine levels of TLR4, Dectin-1 as well as downstream inflammatory factors. RESULTS: GLD treatment suppressed the proliferation, biofilm formation abilities and adhesive capability of A. fumigatus. In mice upon A. fumigatus infection, treatment of GLD showed significantly decreased severity of corneal inflammation, reduced number of A. fumigatus in cornea, and suppressed neutrophil infiltration in cornea. GLD treatment obviously inhibited mRNA and protein levels of Dectin-1, TLR4 and proinflammatory mediators such as IL-1ß, HMGB1, and TNF-α in mice corneas compared to the control group. CONCLUSION: GLD has antifungal and anti-inflammatory effects in fungal keratitis through suppressing A. fumigatus proliferation and alleviating neutrophil infiltration, and repressing the expression of TLR4, Dectin-1 and proinflammatory mediators.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway.

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation.

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcAG579R mutation abolishes this interaction. PkcAG579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.

The infectious propagules of Aspergillus fumigatus are coated with antimicrobial peptides.

Fungal spores are unique cells that mediate dispersal and survival in the environment. For pathogenic fungi encountering a susceptible host, these specialised structures may serve as infectious particles. The main causative agent of the opportunistic disease aspergillosis, Aspergillus fumigatus, produces asexual spores, the conidia, that become dissipated by air flows or water currents but also serve as propagules to infect a susceptible host. We demonstrate that the defX gene of this mould encodes putative antimicrobial peptides resembling cysteine-stabilised (CS)αß defensins that are expressed in a specific spatial and temporal manner in the course of asexual spore formation. Localisation studies on strains expressing a fluorescent proxy or tagged defX alleles expose that these antimicrobial peptides are secreted to coat the conidial surface. Deletion mutants reveal that the spore-associated defX gene products delay the growth of Gram-positive Staphylococcus aureus and demonstrate that the defX gene and presumably its encoded spore-associated defensins confer a growth advantage to the fungal opponent over bacterial competitors. These findings have implications with respect to the ecological niche of A. fumigatus that serves as a 'virulence school' for this human pathogenic mould; further relevance is given for the infectious process resulting in aspergillosis, considering competition with the host microbiome or co-infecting microorganisms to break colonisation resistance at host surfaces.

Influenza Suppresses Neutrophil Recruitment to the Lung and Exacerbates Secondary Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus is an environmental fungus that can cause invasive pulmonary aspergillosis when spores are inhaled into the respiratory tract and invade airway or lung tissue. Influenza is a common respiratory virus that can cause severe respiratory disease, and postinfluenza invasive pulmonary aspergillosis, which is becoming a well-recognized clinical problem, typically occurs in critically ill patients. Mice challenged with influenza A PR/8/34 H1N1 and subsequently challenged with A. fumigatus had increased fungal burden, viral burden, inflammation, and mortality compared with single infected mice. Neutrophil recruitment in the lung of superinfected mice was decreased; however, mice were not neutropenic, and there was no difference in absolute blood neutrophils between groups. Additionally, CXCL1 and CXCL2 were decreased in lungs of superinfected mice compared with controls. IFN levels were increased in mice that received influenza, and deletion of STAT1 resulted in decreased fungal burden, increased airway and lung neutrophils, and increased CXCL1 compared with wild-type mice, whereas deletion of STAT2 did not change fungal burden or airway neutrophilia compared with wild-type mice. These data demonstrate a mechanism by which influenza A-induced STAT1 signaling inhibits neutrophil recruitment and increases susceptibility to postinfluenza invasive pulmonary aspergillosis.

Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation.

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI-H520 squamous cells increased intracellular calcium levels and granulocyte macrophage-colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air-liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1ß, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

Invasive pulmonary aspergillosis in the COVID-19 era: An expected new entity.

OBJECTIVES: Information on the recently COVID-19-associated pulmonary aspergillosis (CAPA) entity is scarce. We describe eight CAPA patients, compare them to colonised ICU patients with coronavirus disease 2019 (COVID-19), and review the published literature from Western countries. METHODS: Prospective study (March to May, 2020) that included all COVID-19 patients admitted to a tertiary hospital. Modified AspICU and European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria were used. RESULTS: COVID-19-associated pulmonary aspergillosis was diagnosed in eight patients (3.3% of 239 ICU patients), mostly affected non-immunocompromised patients (75%) with severe acute respiratory distress syndrome (ARDS) receiving corticosteroids. Diagnosis was established after a median of 15 days under mechanical ventilation. Bronchoalveolar lavage was performed in two patients with positive Aspergillus fumigatus cultures and galactomannan (GM) index. Serum GM was positive in 4/8 (50%). Thoracic CT scan findings fulfilled EORTC/MSG criteria in one case. Isavuconazole was used in 4/8 cases. CAPA-related mortality was 100% (8/8). Compared with colonised patients, CAPA subjects were administered tocilizumab more often (100% vs. 40%, p = .04), underwent longer courses of antibacterial therapy (13 vs. 5 days, p = .008), and had a higher all-cause mortality (100% vs. 40%, p = .04). We reviewed 96 similar cases from recent publications: 59 probable CAPA (also putative according modified AspICU), 56 putative cases and 13 colonisations according AspICU algorithm; according EORTC/MSG six proven and two probable. Overall, mortality in the reviewed series was 56.3%. CONCLUSIONS: COVID-19-associated pulmonary aspergillosis must be considered a serious and potentially life-threatening complication in patients with severe COVID-19 receiving immunosuppressive treatment.

A Shifted Composition of the Lung Microbiota Conditions the Antifungal Response of Immunodeficient Mice.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1-/- mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells.

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.

Aspergillus fumigatus and Aspergillosis in 2019.

Aspergillus fumigatus is a saprotrophic fungus; its primary habitat is the soil. In its ecological niche, the fungus has learned how to adapt and proliferate in hostile environments. This capacity has helped the fungus to resist and survive against human host defenses and, further, to be responsible for one of the most devastating lung infections in terms of morbidity and mortality. In this review, we will provide (i) a description of the biological cycle of A. fumigatus; (ii) a historical perspective of the spectrum of aspergillus disease and the current epidemiological status of these infections; (iii) an analysis of the modes of immune response against Aspergillus in immunocompetent and immunocompromised patients; (iv) an understanding of the pathways responsible for fungal virulence and their host molecular targets, with a specific focus on the cell wall; (v) the current status of the diagnosis of different clinical syndromes; and (vi) an overview of the available antifungal armamentarium and the therapeutic strategies in the clinical context. In addition, the emergence of new concepts, such as nutritional immunity and the integration and rewiring of multiple fungal metabolic activities occurring during lung invasion, has helped us to redefine the opportunistic pathogenesis of A. fumigatus.

Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing.

In immunocompromised individuals, Aspergillus fumigatus causes invasive fungal disease that is often difficult to treat. Exactly how immune mechanisms control A. fumigatus in immunocompetent individuals remains unclear. Here, we use transparent zebrafish larvae to visualize and quantify neutrophil and macrophage behaviors in response to different A. fumigatus strains. We find that macrophages form dense clusters around spores, establishing a protective niche for fungal survival. Macrophages exert these protective effects by inhibiting fungal germination, thereby inhibiting subsequent neutrophil recruitment and neutrophil-mediated killing. Germination directly drives fungal clearance as faster-growing CEA10-derived strains are killed better in vivo than slower-growing Af293-derived strains. Additionally, a CEA10 pyrG-deficient strain with impaired germination is cleared less effectively by neutrophils. Host inflammatory activation through Myd88 is required for killing of a CEA10-derived strain but not sufficient for killing of an Af293-derived strain, further demonstrating the role of fungal-intrinsic differences in the ability of a host to clear an infection. Altogether, we describe a new role for macrophages in the persistence of A. fumigatus and highlight the ability of different A. fumigatus strains to adopt diverse modes of virulence.

The Transcriptional Regulator HbxA Governs Development, Secondary Metabolism, and Virulence in Aspergillus fumigatus.

Aspergillus fumigatus is the leading cause of invasive aspergillosis, which in immunocompromised patients results in a mortality rate as high as 90%. Earlier studies showed that HbxA is a global regulator in Aspergillus flavus affecting morphological development and secondary metabolism. Here, we determined its role in A. fumigatus, examining whether HbxA influences the regulation of asexual development, natural product biosynthesis, and virulence of this fungus. Our analysis demonstrated that removal of the hbxA gene caused a near-complete loss of conidial production in the mutant strain, as well as a slight reduction in colony growth. Other aspects of asexual development are affected, such as size and germination of conidia. Furthermore, we showed that in A. fumigatus, the loss of hbxA decreased the expression of the brlA central regulatory pathway involved in asexual development, as well as the expression of the "fluffy" genes flbB, flbD, and fluG HbxA was also found to regulate secondary metabolism, affecting the biosynthesis of multiple natural products, including fumigaclavines, fumiquinazolines, and chaetominine. In addition, using a neutropenic mouse infection model, hbxA was found to negatively impact the virulence of A. fumigatusIMPORTANCE The number of immunodepressed individuals is increasing, mainly due to the greater life expectancy in immunodepressed patients due to improvements in modern medical treatments. However, this population group is highly susceptible to invasive aspergillosis. This devastating illness, mainly caused by the fungus Aspergillus fumigatus, is associated with mortality rates reaching 90%. Treatment options for this disease are currently limited, and a better understanding of A. fumigatus genetic regulatory mechanisms is paramount for the design of new strategies to prevent or combat this infection. Our work provides new insight into the regulation of the development, metabolism, and virulence of this important opportunistic pathogen. The transcriptional regulatory gene hbxA has a profound effect on A. fumigatus biology, governing multiple aspects of conidial development. This is relevant since conidia are the main source of inoculum in Aspergillus infections. Importantly, hbxA also regulates the biosynthesis of secondary metabolites and the pathogenicity of this fungus.