Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures

Abstract

Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8⁺ IL-17⁺ (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia.     

Monoclonal Immunoglobulin G1 Directed against Aspergillus fumigatus Cell Wall Glycoprotein Protects against Experimental Murine Aspergillosis

Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log₁₀ units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 × 10⁵ CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.     

The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.     

Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Previously, we identified a mutant strain (white, W) lacking conidial pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to oxidants in vitro and more efficiently damaged by human monocytes in vitro than WT conidia. When compared to the WT, the W mutant strain showed reduced virulence in a murine animal model. Genetic analysis suggested that the W mutant carried a single mutation which caused all of the observed phenotypes. Here, we report the construction of a genomic cosmid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transformation with plasmid pHELP1 carrying the autonomously replicating ama1 sequence of A. nidulans which also increased the transformation efficiency of A. fumigatus by a factor of 10. Using this cosmid library a putative polyketide synthase gene, designated pksP (polyketide synthase involved in pigment biosynthesis) was isolated. The pksP gene has a size of 6660 bp. pksP consists of five exons separated by short (47–73 bp) introns. Its deduced open reading frame is composed of 2146 amino acids. The pksP gene complemented both the white phenotype and the surface morphology of the W mutant conidia to wild type. Whereas W mutant conidia caused a strong reactive oxygen species (ROS) release by polymorphonuclear leukocytes, the ability of pksP-complemented W mutant conidia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed restored virulence in a mouse model. Received: 10 August 1998     

Antioxidant systems in the pathogenic fungi of man and their role in virulence.

In the last two decades, a variety of fungal antioxidants have attracted considerable interest, largely arising from their hypothetical role as virulence determinants. Melanin is a potent free radical scavenger and in Cryptococcus neoformans, there is now good evidence that the production of melanin is a significant virulence determinant. There is also recent evidence linking melanin biosynthesis to the virulence of Aspergillus fumigatus conidia. Superoxide dismutases are important housekeeping antioxidants and have an additional hypothetical role in virulence; however, although these enzymes have been biochemically characterized from Aspergillus and Cryptococcus, there is as yet no firm evidence that these enzymes are involved in pathogenicity. Catalase production may play some role in the virulence of Candida albicans but this enzyme has not been shown, as yet, to influence the virulence of A. fumigatus. There are some data supporting an antioxidant function for the acyclic hexitol mannitol in C. neoformans, but further investigations are required in this area. Research into the putative antioxidant activities of a range of other fungal enzymes, such as acid phosphatases, remains limited at this time.     

Biosynthesis and functions of melanin in Sporothrix schenckii

Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.     

Early Events in Macrophage Killing of Aspergillus fumigatus Conidia: New Flow Cytometric Viability Assay

Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.     

Production of melanin pigments in saprophytic fungi in vitro and during infection

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin‐specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin‐specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.     

Comparative Study of Susceptibilities of Germinated and Ungerminated Conidia of Aspergillus fumigatus to Various Antifungal Agents

Conidia are used as inocula for the in vitro susceptibility testing of Aspergillus fumigatus. Since the MIC is defined on the basis of visible mycelial growth, conidia should germinate and produce sporelings (germinated conidia) for monitoring of the growth inhibition and fungicidal activity of a drug. If a compound is capable of inhibiting germination of conidia while affecting or not affecting the growth of the organism, the MIC obtained will be the concentration of the drug required for the inhibition of conidial germination but not necessarily that required for inhibition of the growth of the organism. We investigated the susceptibility of germinated and ungerminated conidia to amphotericin B, itraconazole, voriconazole, and SCH56592. The MICs of various antifungal agents for germinated conidia were almost identical to those obtained for ungerminated conidia. In addition, both the germinated and ungerminated conidia were killed with almost equal efficiency by all of the compounds tested when exposed to the drugs for 24 h. These results suggest that either germinated or ungerminated conidia could be used as inocula for in vitro susceptibility studies of A. fumigatus with identical results.     

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia,but is dispensable for pathogenicity in Magnaporthe oryzae

Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.     

Secreted Aspergillus fumigatus Protease Alp1 Degrades Human Complement Proteins C3, C4, and C5

The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack.Aspergillus fumigatus is the most important airborne fungal pathogen. The frequency of invasive mycoses due to this opportunistic fungal pathogen has increased significantly during the last 2 decades (reviewed in references 7 and 42). In healthy individuals, A. fumigatus conidia are inhaled but the establishment of disease is prevented by the host immune system. Inhaled A. fumigatus conidia are immediately confronted with the host complement system and phagocytic cells. The complement system is activated on the conidial and hyphal surface (26), and this activation results in the cleavage of C3. Cleavage products of this central component of the complement cascade act as opsonins on the surfaces of pathogens and enhance phagocytosis by neutrophils, macrophages, and eosinophils (69). Opsonization with complement proteins is important for phagocytosis of A. fumigatus conidia, the key process in the defense against this pathogen (59).Activation of the complement system occurs via three pathways: the alternative pathway (AP), the lectin pathway, and the classical pathway. The AP is activated on microbial surfaces and plays a pivotal role in the clearance of microorganisms (70). Progression of the cascade leads to generation of a C5 convertase, which produces inflammatory C5a anaphylatoxins, and also to the formation of terminal complement complexes (TCC), which can form membrane attack complexes (MAC) and hence pores on target surfaces. C3b surface deposition and MAC formation are important for clearance of bacteria but appear to play a minor role in the defense against fungi. The complement activation system is controlled by fluid-phase and cell surface-bound regulators. We and others showed before (2, 62) that A. fumigatus conidia bind factor H (the central human regulator of the AP), FHL-1, and CFHR1. Factor H acts as a cofactor for the plasma serine protease factor I, which mediates the cleavage of C3b (21, 35, 41). This blocks C3 convertase formation and leads to downregulation or termination of the complement cascade. In contrast to conidia, A. fumigatus hyphae do not bind factor H (2). Instead, they activate complement on their surface (26). Until now a single, nonprotein activity in culture supernatant that inhibits opsonization of the fungal surface by complement proteins has been described (65). The nature of this molecule has not been discovered yet. A. fumigatus inhibition of complement activation is important, since activation of the complement cascade causes toxic and damaging effector functions. Although hyphae are too big to become phagocytosed by macrophages, attraction and activation of neutrophils by C3a and C5a still lead to the destruction of hyphae. Therefore, here we analyzed whether A. fumigatus hyphae use additional strategies to interfere with the human complement system.     

Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.     

T-2 toxin impairs antifungal activities of chicken macrophages against Aspergillus fumigatus conidia but promotes the pro-inflammatory responses

Aspergillosis is the most common fungal disease of the avian respiratory tract and is caused primarily by Aspergillus fumigatus. The respiratory macrophages provide important defence against aspergillosis. T-2 toxin (T-2), a trichothecene mycotoxin produced by Fusarium spp. in improperly stored agricultural products, has immunomodulatory effects. We studied the impact of T-2 on the antifungal response of the chicken macrophage cell line HD-11 against A. fumigatus infection. The macrophages were first exposed to 0.5 to 10 ng/ml T-2 for 24 h, and then their viability, antifungal activity, and cytokine expression in response to A. fumigatus conidial infection were determined. The viability of macrophages decreased when exposed to T-2 at concentrations higher than 1 ng/ml. One hour after conidial infection, phagocytosed conidia were observed in 30% of the non-T-2-exposed macrophages, but in only 5% of the macrophages exposed to 5 ng/ml T-2. Seven hours after infection, 24% of the conidia associated with non-T-2-exposed macrophages germinated, in contrast to 75% of those with macrophages exposed to 5 ng/ml T-2. A. fumigatus infection induced upregulation of interleukin (IL)-1β, CXCLi1, CXCLi2 and IL-12β, and downregulation of transforming growth factor-β4 in macrophages. Exposure of A. fumigatus-infected macrophages to T-2 at 1 to 5 ng/ml further upregulated the expression of IL-1β, IL-6, CCLi2, CXCLi1, CXCLi2, IL-18 (at 1 and 2 ng/ml) and IL-12β, and further downregulated that of transforming growth factor-β4 (at 5 ng/ml). In conclusion, T-2 impaired the antifungal activities of chicken macrophages against A. fumigatus conidia, but might stimulate immune response by upregulating the expression of pro-inflammatory cytokines, chemokines and T-helper 1 cytokines.     

Effect of Melanin and Carotenoids of Exophiala (Wangiella) dermatitidis on Phagocytosis, Oxidative Burst, and Killing by Human Neutrophils

The black yeast Exophiala (Wangiella) dermatitidis is an increasingly recognized pathogen and a leading cause of severe pheohyphomycosis. Melanin is thought to contribute to the virulence of E. dermatitidis. Whereas the synthesis and the redox properties of melanin have been studied intensively, the influence of melanin and carotenoids on the phagocytosis, the oxidative burst, and the killing of E. dermatitidis by human neutrophils has not been studied. To study their effects on these phenomena, we applied a combination of flow cytometry and a colony-count-dependent method. Using E. dermatitidis wild-type strain 8565 and several melanin-deficient mutants that have been described previously, we demonstrate that melanin prevents this pathogen from being killed in the phagolysosome of the neutrophils. Melanin did not influence the phagocytosis or the oxidative burst of the neutrophils involved. The carotenoids torulene and torularhodine were not found to contribute to the prevention of killing. The ability of E. dermatitidis to block the effects of the neutrophil oxidative burst may critically impair the potential of the host to sufficiently eliminate this fungal pathogen and thus may play an important role in the pathogenesis of phaeohyphomycosis.     

Cochliobolus lunatus colonizes potato by adopting different invasion strategies on cultivars: New insights on temperature dependent-virulence

Extreme temperature fluctuations affect the interaction dynamics of Cochliobolus lunatus through temperature-dependent virulence, virulence differentiation and induced-virulence which poses a major threat to global food security. The relationship between higher temperature and pathogenicity of C. lunatus on reported hosts are poorly understood. In this study, temperature stress was applied on C. lunatus to investigate the correlation among the different types of conidia. Additionally, a comparative dissection of the invasion process, infection structures and conidial germination pattern on four different Solanum tuberosum L. (potato) cultivars were performed. Based on microscopic examination, it was found that C. lunatus adopts different hyphae morphology and septation pattern at different temperature regimes and produce different types of conidia. The study showed that four-celled conidia are overproduced at elevated temperature (>30 °C) than one, two, three and five-celled conidia. Our finding revealed that C. lunatus conidia exhibit bipolar germination (>14.67%, P < 0.05), unipolar germination (>35.33%, P < 0.05), penetrate subcutaneously via epidermal anticlinal cell wall (>0.33%, P < 0.05) and differentially form appressoria-like structures during colonization of four different potato cultivars. Importantly, it is shown that unipolar germination and bipolar germination in C. lunatus are independently occurring phenomenon irrespective of the host. It is confirmed that C. lunatus adopt different but highly successful strategies on four different potato cultivars to incite brown-to-black leaf spot disease. Altogether, our data showed that increase in temperature enhances C. lunatus virulence on different potato cultivars irrespective of their inherent thermotolerant traits.     

AfMkk2 is required for cell wall integrity signaling,adhesion, and full virulence of the human pathogen Aspergillus fumigatus

The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.     

Melanin from Fonsecaea pedrosoi induces production of human antifungal antibodies and enhances the antimicrobial efficacy of phagocytes

Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients' sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients' lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.     

Adoptive immunity mediated by HLA-A*0201 restricted Asp f16 peptides-specific CD8+ T cells against Aspergillus fumigatus infection

Aspergillus fumigatus (A. fumigatus) is the most common pathogen of invasive aspergillosis (IA), a life-threatening infection in immunocompromised patients. Recent findings revealed that CD8+ T cells can mediate cytotoxic activity against A. fumigatus. Here, we bioinformatically identified three HLA-A*0201-restricted peptides from Asp f16, an A. fumigatus antigen which was previously shown to be involved in T cell immunity. Our immunological results demonstrated that these peptides can potently induce cytotoxic T lymphocyte (CTL) response in CD8+ T cells, thus, damaging the conidia and hyphae of A. fumigatus. Moreover, the Asp f16 peptides can also raise Th1 cell-like response, as measured by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT). Furthermore, we established an invasive pulmonary aspergillosis model in HLA-A*0201 transgenic mice. Adoptive transfer of Asp f16 peptides-specific CTL significantly extended the overall survival time in the A. fumigatus-infected immunocompromised mice. In conclusion, our results demonstrate that the Asp f16 peptides might provide immunity against invasive A. fumigatus infection.