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Caspofungin, a lipopeptide, is an antifungal drug that belong to the class of echinocandin. It inhibits fungal cell wall β-(1,3)-glucan synthase activity and is the second-line of drug for invasive aspergillosis, a fatal infection caused mainly by Aspergillus fumigatus. On the other hand, Enfumafungin is a natural triterpene glycoside also with a β-(1,3)-glucan synthase inhibitory activity and reported to have antifungal potential. In the present study, we compared the growth as well as modifications in the A. fumigatus cell wall upon treatment with Caspofungin or Enfumafungin, consequentially their immunomodulatory capacity on human dendritic cells. Caspofungin initially inhibited the growth of A. fumigatus, but the effect was lost over time. By contrast, Enfumafungin inhibited this fungal growth for the duration investigated. Both Caspofungin and Enfumafungin caused a decrease in the cell wall β-(1,3)-glucan content with a compensatory increase in the chitin, and to a minor extent they also affected cell wall galactose content. Treatment with these two antifungals did not result in the exposure of β-(1,3)-glucan on A. fumigatus mycelial surface. Enzymatic digestion suggested a modification of β-(1,3)-glucan structure, specifically its branching, upon Enfumafungin treatment. While there was no difference in the immunostimulatory capacity of antifungal treated A. fumigatus conidia, alkali soluble-fractions from Caspofungin treated mycelia weakly stimulated the dendritic cells, possibly due to an increased content of immunosuppressive polysaccharide galactosaminogalactan. Overall, we demonstrate a novel mechanism that Enfumafungin not only inhibits β-(1,3)-glucan synthase activity, but also causes modifications in the structure of β-(1,3)-glucan in the A. fumigatus cell wall., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Pentraxin 3 (PTX3), a long pentraxin and a humoral pattern recognition molecule (PRM), has been demonstrated to be protective against Aspergillus fumigatus, an airborne human fungal pathogen. We explored its mode of interaction with A. fumigatus, and the resulting implications in the host immune response. Here, we demonstrate that PTX3 interacts with A. fumigatus in a morphotype-dependent manner: (a) it recognizes germinating conidia through galactosaminogalactan, a surface exposed cell wall polysaccharide of A. fumigatus, (b) in dormant conidia, surface proteins serve as weak PTX3 ligands, and (c) surfactant protein D (SP-D) and the complement proteins C1q and C3b, the other humoral PRMs, enhance the interaction of PTX3 with dormant conidia. SP-D, C3b or C1q opsonized conidia stimulated human primary immune cells to release pro-inflammatory cytokines and chemokines. However, subsequent binding of PTX3 to SP-D, C1q or C3b opsonized conidia significantly decreased the production of pro-inflammatory cytokines/chemokines. PTX3 opsonized germinating conidia also significantly lowered the production of pro-inflammatory cytokines/chemokines while increasing IL-10 (an anti-inflammatory cytokine) released by immune cells when compared to the unopsonized counterpart. Overall, our study demonstrates that PTX3 recognizes A. fumigatus either directly or by interplaying with other humoral PRMs, thereby restraining detrimental inflammation. Moreover, PTX3 levels were significantly higher in the serum of patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA), supporting previous observations in IPA patients, and suggesting that it could be a potential panel-biomarker for these pathological conditions caused by A. fumigatus., (© 2024. The Author(s).)

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Heilig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 de Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant., Results: In this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress., Conclusions: Our data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells., (© 2023. The Author(s).)

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides β-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

Invasive aspergillosis (IA) is a life-threatening fungal infection for immunocompromised hosts. It is, therefore, necessary to understand the immune pathways that control this infection. Although the primary infection site is the lungs, aspergillosis can disseminate to other organs through unknown mechanisms. Herein we have examined the in vivo role of various complement pathways as well as the complement receptors C3aR and C5aR1 during experimental systemic infection by Aspergillus fumigatus , the main species responsible for IA. We show that C3 knockout (C3 -/- ) mice are highly susceptible to systemic infection of A. fumigatus . Intriguingly, C4 -/- and factor B (FB) -/- mice showed susceptibility similar to the wild-type mice, suggesting that either the complement pathways display functional redundancy during infection (i.e., one pathway compensates for the loss of the other), or complement is activated non-canonically by A. fumigatus protease. Our in vitro study substantiates the presence of C3 and C5 cleaving proteases in A. fumigatus . Examination of the importance of the terminal complement pathway employing C5 -/- and C5aR1 -/- mice reveals that it plays a vital role in the conidial clearance. This, in part, is due to the increased conidial uptake by phagocytes. Together, our data suggest that the complement deficiency enhances the susceptibility to systemic infection by A. fumigatus ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shende, Wong, Meitei, Lal, Madan, Aimanianda, Pal and Sahu.)

Hypothesis: Precise modulation of immuno-inflammatory response is crucial to control periodontal diseases and related systemic comorbidities. The present nanosystem with the controlled-release and cell-penetrating manner enhances the inflammation modulation effects of baicalein in human gingival epithelial cells (hGECs) for better oral healthcare., Experiments: We constructed a red-emissive mesoporous silica nanoparticle-based nanosystem with cell-penetrating poly(disulfide) (CPD) capping, through a facile in-situ polymerization approach. It was featured with a glutathione-responsive manner and instant cellular internalization capacity for precisely delivering baicalein intracellularly. Laboratory experiments assessed whether and how the nanosystem per se with the delivered baicalein could modulate immuno-inflammatory responses in hGECs., Findings: The in-situ polymerized CPD layer capped the nanoparticles and yet controlled the release of baicalein in a glutathione-responsive manner. The CPD coating could facilitate cellular internalization of the nanosystem via endocytosis and thiol-mediated approaches. Notably, the intracellularly released baicalein effectively downregulated the expression of pro-inflammatory cytokines through inhibiting the NF-κB signaling pathway. The nanosystem per se could modulate immuno-inflammatory responses by passivating the cellular response to interlukin-1β. This study highlights that the as-synthesized nanosystem may serve as a novel multi-functional vehicle to modulate innate host response via targeting the NF-κB pathway for precision healthcare., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Humoral immunity plays a defensive role against invading microbes. However, it has been largely overlooked with respect to Aspergillus fumigatus , an airborne fungal pathogen. Previously, we have demonstrated that surfactant protein D (SP-D), a major humoral component in human lung-alveoli, recognizes A. fumigatus conidial surface exposed melanin pigment. Through binding to melanin, SP-D opsonizes conidia, facilitates conidial phagocytosis, and induces the expression of protective pro-inflammatory cytokines in the phagocytic cells. In addition to melanin, SP-D also interacts with galactomannan (GM) and galactosaminogalactan (GAG), the cell wall polysaccharides exposed on germinating conidial surfaces. Therefore, we aimed at unravelling the biological significance of SP-D during the germination process. Here, we demonstrate that SP-D exerts direct fungistatic activity by restricting A. fumigatus hyphal growth. Conidial germination in the presence of SP-D significantly increased the exposure of cell wall polysaccharides chitin, α-1,3-glucan and GAG, and decreased β-1,3-glucan exposure on hyphae, but that of GM was unaltered. Hyphae grown in presence of SP-D showed positive immunolabelling for SP-D. Additionally, SP-D treated hyphae induced lower levels of pro-inflammatory cytokine, but increased IL-10 (anti-inflammatory cytokine) and IL-8 (a chemokine) secretion by human peripheral blood mononuclear cells (PBMCs), compared to control hyphae. Moreover, germ tube surface modifications due to SP-D treatment resulted in an increased hyphal susceptibility to voriconazole, an antifungal drug. It appears that SP-D exerts its anti- A. fumigatus functions via a range of mechanisms including hyphal growth-restriction, hyphal surface modification, masking of hyphal surface polysaccharides and thus altering hyphal immunostimulatory properties., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The signaling pathways activated following interaction between dendritic cells (DCs) and a pathogen determine the polarization of effector T-cell and regulatory T-cell (Treg) responses to the infection. Several recent studies, mostly in the context of bacterial infections, have shown that the Wnt/β-catenin pathway plays a major role in imparting tolerogenic features in DCs and in promotion of Treg responses. However, the significance of the Wnt/β-catenin pathway's involvement in regulating the immune response to the fungal species is not known. Using Aspergillus fumigatus, a ubiquitous airborne opportunistic fungal species, we show here that fungi activate the Wnt/β-catenin pathway in human DCs and are critical for mediating the immunosuppressive Treg responses. Pharmacological inhibition of this pathway in DCs led to inhibition of maturation-associated molecules and interleukin 10 (IL-10) secretion without affecting the majority of the inflammatory cytokines. Furthermore, blockade of Wnt signaling in DCs suppressed DC-mediated Treg responses in CD4 + T cells and downregulated both tumor necrosis factor alpha (TNF-α) and IL-10 responses in CD8 + T cells. Mechanistically, induction of β-catenin pathway by A. fumigatus required C-type lectin receptors and promoted Treg polarization via the induction of programmed death-ligand 1 on DCs. Further investigation on the identity of fungal molecular patterns has revealed that the cell wall polysaccharides β-(1, 3)-glucan and α-(1, 3)-glucan, but not chitin, possess the capacity to activate the β-catenin pathway. Our data suggest that the Wnt/β-catenin pathway is a potential therapeutic target to selectively suppress the Treg response and to sustain the protective Th1 response in the context of invasive aspergillosis caused by A. fumigatus. IMPORTANCE The balance between effector CD4 + T-cell and immunosuppressive regulatory T-cell (Treg) responses determines the outcome of an infectious disease. The signaling pathways that regulate human CD4 + T-effector versus Treg responses to the fungi are not completely understood. By using Aspergillus fumigatus, a ubiquitous opportunistic fungal species, we show that fungi activate the Wnt/β-catenin pathway in human dendritic cells (DCs) that promotes Treg responses via induction of immune checkpoint molecule programmed death ligand 1 on DCs. Blockade of the Wnt/β-catenin pathway in DCs led to the selective inhibition of Treg without affecting the Th1 response. Dissection of the identity of A. fumigatus pathogen-associated molecular patterns (PAMPs) revealed that cell wall polysaccharides exhibit selectivity in their capacity to activate the β-catenin pathway in DCs. Our data thus provide a pointer that Wnt/β-catenin pathway represents potential therapeutic target to selectively suppress Treg responses and to sustain protective a Th1 response against invasive fungal diseases.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii , two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii , suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Neves, Wong, Aimanianda, Simenel, Guijarro, Walls, Willment, Gow, Munro, Brown and Lopes-Bezerra.)

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii . Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Humoral immune components have been individually studied in the context of interaction of host with Aspergillus fumigatus , a major airborne fungal pathogen. However, a global view of the multitude and complex nature of humoral immune components is needed to bring new insight into host- Aspergillus interaction. Therefore, we undertook comparative proteomic analysis of the bronchoalveolar lavage fluid collected from individuals infected or colonized with A. fumigatus versus controls, to identify those alveolar humoral components affected upon A. fumigatus infection. Complement proteins C1q, C8 beta-chain, factor-H, ficolin-1, ficolin-2, mannan binding lectin serine peptidase 2, pentraxin-3 and the surfactant protein-D were identified as the major humoral immune components affected by A. fumigatus infection and colonization. Based on this observation, we hypothesize that crosstalk between these humoral components is essential during host- Aspergillus interaction giving new specific leads to study for better understanding the pathogenesis. Furthermore, the affected humoral components could be potential diagnostic markers of A. fumigatus infection or colonization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dellière, Duchateau, Wong, Giai Gianetto, Guegan, Matondo, Gangneux and Aimanianda.)

Although belong to the same genus, Aspergillus fumigatus is primarily involved in invasive pulmonary infection, whereas Aspergillus flavus is a common cause of superficial infection. In this study, we compared conidia (the infective propagules) of these two Aspergillus species. In immunocompetent mice, intranasal inoculation with conidia of A. flavus resulted in significantly higher inflammatory responses in the lungs compared to mice inoculated with A. fumigatus conidia. In vitro assays revealed that the dormant conidia of A. flavus , unlike A. fumigatus dormant conidia, are immunostimulatory. The conidial surface of A. fumigatus was covered by a rodlet-layer, while that of A. flavus were presented with exposed polysaccharides. A. flavus harbored significantly higher number of proteins in its conidial cell wall compared to A. fumigatus conidia. Notably, β-1,3-glucan in the A. flavus conidial cell-wall showed significantly higher percentage of branching compared to that of A. fumigatus . The polysaccharides ensemble of A. flavus conidial cell wall stimulated the secretion of proinflammatory cytokines, and conidial cell wall associated proteins specifically stimulated IL-8 secretion from the host immune cells. Furthermore, the two species exhibited different sensitivities to antifungal drugs targeting cell wall polysaccharides, proposing the efficacy of species-specific treatment strategies. Overall, the species-specific organization of the conidial cell wall could be important in establishing infection by the two Aspergillus species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wong, Venugopalan, Beaussart, Karnam, Mohammed, Jayapal, Bretagne, Bayry, Prajna, Kuppamuthu, Latgé and Aimanianda.)

Candida albicans is a commensal polymorphic and opportunistic fungus, which usually resides as a small community in the oral cavities of a majority of humans. The latter eco-system presents this yeast varied opportunities for mutualistic interactions with other cohabitant oral bacteria, that synergizes its persistence and pathogenicity. Collectively, these communities live within complex plaque biofilms which may adversely affect the oral health and increase the proclivity for oral candidiasis. The proteome of such oral biofilms with myriad interkingdom interactions are largely underexplored. Herein, we employed limma differential expression analysis, and cluster analysis to explore the proteomic interactions of C. albicans biofilms with nine different common oral bacterial species, Aggregatibacter actinomycetemcomitans , Actinomyces naeslundii , Fusobacterium nucleatum , Enterococcus faecalis , Porphyromonas gingivalis , Streptococcus mutants , Streptococcus sanguinis , Streptococcus mitis , and Streptococcus sobrinus . Interestingly, upon exposure of C. albicans biofilms to the foregoing heat-killed bacteria, the proteomes of the fungus associated with cellular respiration, translation, oxidoreductase activity, and ligase activity were significantly altered. Subsequent differential expression and cluster analysis revealed the subtle, yet significant alterations in the C. albicans proteome, particularly on exposure to bacteria with dissimilar cell morphologies, and Gram staining characteristics.

Immune inertness of Aspergillus fumigatus conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp ) mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the RODA deletion mutant (∆ rodA ). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp - mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆ rodA conidia selectively bound to β-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆ rodA was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆ rodA . Ccrp-mutant conidia were less immunostimulatory compared to ∆ rodA conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host-fungal interaction., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, β-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche., (Copyright © 2020 American Society for Microbiology.)

Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the c aspofungin p aradoxical e ffect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The Δ fhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the Δ fhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance. IMPORTANCE Aspergillus fumigatus , one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus , but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the c aspofungin p aradoxical e ffect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance., (Copyright © 2020 Valero et al.)

In humans, loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3) gene is frequently associated with susceptibility to bacterial as well as fungal infections including aspergillosis, although its pathogenesis remains largely unknown. In the present study, we investigated the immune responses obtained after stimulation with Aspergillus fumigatus in STAT3-deficient patients. A. fumigatus conidial killing efficiencies of both monocytes and neutrophils isolated from whole blood samples of STAT3-deficient patients were not different compared to those of healthy controls. After stimulation with A. fumigatus conidia, lower concentrations of adaptive cytokines (IFN-γ, IL-17 and IL-22) were secreted by peripheral blood mononuclear cells from STAT3-deficient patients compared to those from healthy controls. Moreover, the frequency of IFN-γ and IL-17 producing CD4+ T cells was lower in STAT3-deficient patients vs. healthy controls. Among the STAT3-deficient patients, those with aspergillosis showed further lower secretion of IFN-γ upon stimulation of their PBMCs with A. fumigatus conidia compared to the patients without aspergillosis. Together, our study indicated that STAT3-deficiency leads to a defective adaptive immune response against A. fumigatus infection, particularly with a lower IFN-γ and IL-17 responses in those with aspergillosis, suggesting potential therapeutic benefit of recombinant IFN-γ in STAT3-deficient patients with aspergillosis., (Copyright © 2020 Danion, Aimanianda, Bayry, Duréault, Wong, Bougnoux, Tcherakian, Alyanakian, Guegan, Puel, Picard, Lortholary, Lanternier and Latgé.)

Methodologies to identify epitopes or ligands of the fungal cell wall polysaccharides influencing the immune response of human pathogens have to date been imperfect. Using the galactomannan (GM) of Aspergillus fumigatus as a model, we have shown that synthetic oligosaccharides of distinct structures representing key fragments of cell wall polysaccharides are the most precise tools to study the serological and immunomodulatory properties of a fungal polysaccharide., (Copyright © 2020 Wong et al.)

Oral candidiasis as a highly prevalent and recurrent infection in medically compromised individuals is mainly caused by the opportunistic fungal pathogen Candida albicans . This epithelial infection, if not controlled effectively, can progress to life-threatening systemic conditions and complications. The efficacy of current frontline antifungals is limited due to their poor bioavailability and systemic toxicity. As such, an efficient intervention is essential for controlling disease progression and recurrence. Herein, a theranostic nanoplatform (CD-Gu + -AmB) was developed to track the penetration of antifungals and perturb the invasion of C. albicans at oral epithelial tissues, via decorating the homemade red-emissive carbon dots (CD) with positively charged guanidine groups (Gu + ) followed by conjugation with antifungal polyene (amphotericin B, AmB) in a reacting site-controllable manner. The generated CD-Gu + -AmB favorably gathered within the Candida cells and exhibited potent antifungal effects in both planktonic and biofilm forms. It selectively accumulated in the nuclei of human oral keratinocytes and exhibited undetectable toxicity to the host cells. Moreover, we reported for the first time the penetration and exfoliation profiles of CD in a three-dimensional organotypic model of human oral epithelial tissues, demonstrating that the extra- and intracellular accumulation of CD-Gu + -AmB effectively resisted the invasion of C. albicans by forming a "shielding" layer throughout the entire tissue. This study establishes a multifunctional CD-based theranostic nanoplatform functioning as a traceable and topically applied antifungal to arm oral epithelia, thereby shedding light on early intervention of mucosal candidiasis for oral and general health.

β-(1,3)-Glucan is one of the antigenic components of the bacterial as well as fungal cell wall. We designed microcapsules (MCs) ligated with β-(1,3)-glucan, to study its immunomodulatory effect. The MCs were obtained by interfacial polycondensation between diacyl chloride (sebacoyl chloride and terephtaloyl chloride) and diethylenetriamine in organic and aqueous phases, respectively. Planar films were first designed to optimize monomer compositions and to examine the kinetics of film formation. MCs with aqueous fluorescent core were then obtained upon controlled emulsification-polycondensation reactions using optimized monomer compositions and adding fluorescein into the aqueous phase. The selected MC-formulation was grafted with Curdlan, a linear β-(1,3)-glucan from  Agrobacterium species or branched β-(1,3)-glucan isolated from the cell wall of Aspergillus fumigatus. These β-(1,3)-glucan grafted MCs were phagocytosed by human monocyte-derived macrophages, and stimulated cytokine secretion. Moreover, the blocking of dectin-1, a β-(1,3)-glucan recognizing receptor, did not completely inhibit the phagocytosis of these β-(1,3)-glucan grafted MCs, suggesting the involvement of other receptors in the recognition and uptake of β-(1,3)-glucan. Overall, grafted MCs are a useful tool for the study of the mechanism of phagocytosis and immunomodulatory effect of the microbial polysaccharides.

β-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-β-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-β-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing β-glucans with different lengths of oligo-β-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a β-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-β-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these β-glucan-specific mAbs could be useful in combinatorial antifungal therapy., Competing Interests: The authors have declared that no competing interests exist.

Biotinylated hepta-, nona- and undeca-α-(1 → 3)-d-glucosides representing long oligosaccharides of α-(1 → 3)-d-glucan, one of the major components of the cell walls of the fungal pathogen Aspergillus fumigatus, were synthesized for the first time via a blockwise strategy. Convergent assembly of the α-(1 → 3)-d-glucan chains was achieved by glycosylation with oligoglucoside derivatives bearing 6- O-benzoyl groups. Those groups are capable of remote α-stereocontrolling participation, making them efficient α-directing tools even in the case of large glycosyl donors. Synthetic biotinylated oligoglucosides (and biotinylated derivatives of previously synthesized tri- and penta-α-(1 → 3)-d-glucosides) loaded on streptavidin microtiter plates were shown to be better recognized by anti-α-(1 → 3)-glucan human polyclonal antibodies and to induce higher cytokine responses upon stimulation of human peripheral blood mononuclear cells than their natural counterpart, α-(1 → 3)-d-glucan, immobilized on a conventional microtiter plate. Attachment of the synthetic oligosaccharides equipped with a hydrophilic spacer via the streptavidin-biotin pair allows better spatial presentation and control of the loading compared to the random sorption of natural α-(1 → 3)-glucan. Increase of oligoglucoside length results in their better recognition and enhancement of cytokine production. Thus, using synthetic α-(1 → 3)-glucan oligosaccharides, we developed an assay for the host immune response that is more sensitive than the assay based on native α-(1 → 3)-glucan.

Innate immunity in animals including humans encompasses the complement system, which is considered an important host defense mechanism against Aspergillus fumigatus , one of the most ubiquitous opportunistic human fungal pathogens. Previously, it has been shown that the alkaline protease Alp1p secreted from A. fumigatus mycelia degrades the complement components C3, C4, and C5. However, it remains unclear how the fungal spores ( i.e. conidia) defend themselves against the activities of the complement system immediately after inhalation into the lung. Here, we show that A. fumigatus conidia contain a metalloprotease Mep1p, which is released upon conidial contact with collagen and inactivates all three complement pathways. In particular, Mep1p efficiently inactivated the major complement components C3, C4, and C5 and their activation products (C3a, C4a, and C5a) as well as the pattern-recognition molecules MBL and ficolin-1, either by directly cleaving them or by cleaving them to a form that is further broken down by other proteases of the complement system. Moreover, incubation of Mep1p with human serum significantly inhibited the complement hemolytic activity and conidial opsonization by C3b and their subsequent phagocytosis by macrophages. Together, these results indicate that Mep1p associated with and released from A. fumigatus conidia likely facilitates early immune evasion by disarming the complement defense in the human host., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus , but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus , galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D-opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D -/- mice challenged intranasally with wildtype conidia or melanin ghosts ( i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aspergillus fumigatus produce airborne spores (conidia), which are inhaled in abundant quantity. In an immunocompromised population, the host immune system fails to clear the inhaled conidia, which then germinate and invade, leading to pulmonary aspergillosis. In an immunocompetent population, the inhaled conidia are efficiently cleared by the host immune system. Soluble mediators of the innate immunity, that involve the complement system, acute-phase proteins, antimicrobial peptides and cytokines, are often considered to play a complementary role in the defense of the fungal pathogen. In fact, the soluble mediators are essential in achieving an efficient clearance of the dormant conidia, which is the morphotype of the fungus upon inhalation by the host. Importantly, harnessing the host soluble mediators challenges the immunological inertness of the dormant conidia due to the presence of the rodlet and melanin layers. In the review, we summarized the major soluble mediators in the lung that are involved in the recognition of the dormant conidia. This knowledge is essential in the complete understanding of the immune defense against A. fumigatus ., Competing Interests: The authors declare no conflict of interest.

Cyclosporin A (CsA) is widely used as an immunosuppressive agent for organ transplant recipients. CsA inhibits calcineurin, which is highly conserved in mammals and fungi, and thus affects both types of organism. In mammals, the immunosuppressive effect of CsA is via hampering T cell activation. In fungi, the growth inhibitory effect of CsA is via interference with hyphal growth. The aim of this study was to determine whether CsA renders mice susceptible to invasive pulmonary aspergillosis (IPA) and whether it can protect immunosuppressed mice from infection. We therefore examined both the antifungal and the immunosuppressive activity of CsA in immunosuppressed and in immunocompetent mice infected with Aspergillus fumigatus to model IPA. We found that daily injections of CsA could not produce an antifungal effect sufficient to rescue immunosuppressed mice from lethal IPA. However, a 100% survival rate was obtained in non-immunosuppressed mice receiving daily CsA, indicating that CsA did not render the mice vulnerable to IPA. The lymphocyte subset was significantly suppressed by CsA, while the myeloid subset was not. Therefore, we speculate that CsA does not impair the host defense against IPA since the myeloid cells are preserved.

Candida infections have created a great burden on the public healthcare sector. The situation is worsened by recent epidemiological changes. Furthermore, the current arsenal of antifungal agents is limited and associated with undesirable drawbacks. Therefore, new antifungal agents that surpass the existing ones are urgently needed. High-throughput screening of small molecule libraries enables rapid hit identification and, possibly, increases hit rate. Moreover, the identified hits could be associated with unrecognized or multiple drug targets, which would provide novel insights into the biological processes of the pathogen. Hence, it is proposed that high-throughput screening of small molecules is particularly important in the pursuit of the ideal antifungal agents for Candida infections., (Copyright © 2014. Published by Elsevier Ltd.)