Role of Hydrophobins in Aspergillus fumigatus.
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ABSTRACT:
SUBMITTER: Valsecchi I
PROVIDER: S-EPMC5872305 | biostudies-literature | 2017 Dec
REPOSITORIES: biostudies-literature
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Project description:The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and DeltarodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. DeltarodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of DeltarodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the DeltarodA DeltarodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.
| S-EPMC150101 | biostudies-literature
Project description:Copper is an essential metal ion that performs many physiological functions in living organisms. Deletion of Afmac1, which is a copper-responsive transcriptional activator in A. fumigatus, results in a growth defect on aspergillus minimal medium (AMM). Interestingly, we found that zinc starvation suppressed the growth defect of the ?afmac1 strain on AMM. In addition, the growth defect of the ?afmac1 strain was recovered by copper supplementation or introduction of the CtrC gene into the ?afmac1 strain. However, chelation of copper by addition of BCS to AMM failed to recover the growth defect of the ?afmac1 strain. Through Northern blot analysis, we found that zinc starvation upregulated CtrC and CtrA2, which encode membrane copper transporters. Interestingly, we found that the conserved ZafA binding motif 5'-CAA(G)GGT-3' was present in the upstream region of CtrC and CtrA2 and that mutation of the binding motif led to failure of ZafA binding to the upstream region of CtrC and upregulation of CtrC expression under zinc starvation. Furthermore, the binding activity of ZafA to the upstream region of CtrC was inversely proportional to the zinc concentration, and copper inhibited the binding of ZafA to the upstream region of CtrC under a low zinc concentration. Taken together, these results suggest that ZafA upregulates copper metabolism by binding to the ZafA binding motif in the CtrC promoter region under low zinc concentration, thus regulating copper homeostasis. Furthermore, we found that copper and zinc interact in cells to maintain metal homeostasis.
| S-EPMC7593903 | biostudies-literature
Project description:AIM:To investigate the anti-inflammatory role of vasoactive intestinal peptide (VIP) in Aspergillus fumigatus (A. fumigatus) ketatitis. METHODS:Expression of VIP was tested by polymerase chain reaction (PCR) in C57BL/6 and BALB/c normal and A. fumigatus infected corneas. C57BL/6 mice were pretreated with recombinant (r) VIP, while BALB/c mice were pretreated with VIP antagonist, and then infected with A. fumigatus. Clinical score was recorded. Expression of pro- and anti-inflammatory cytokines, toll-like receptor 4 (TLR4), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and neutrophil infiltration were tested by PCR, enzyme-linked immunosorbent assay (ELISA), and myeloperoxidase (MPO) assay. RESULTS:VIP mRNA expression in BALB/c cornea was higher than C57BL/6 cornea at 1 and 3d post infection (p.i.). rVIP treatment of C57BL/6 mice showed alleviated disease and down-regulated expression of interleukin-1? (IL-1?) and tumor necrosis factor-? (TNF-?), while IL-10 expression was up-regulated. Neutrophil infiltration and TLR4, IL-17 expression were decreased after rVIP treatment, while LOX-1 expression was up-regulated in C57BL/6. VIP antagonist pretreatment showed increased disease and higher IL-1?, TNF-?, TLR4, IL-17 and MPO levels, while IL-10 and LOX-1 levels were down-regulated in BALB/c mice. CONCLUSION:rVIP alleviate disease response of C57BL/6 mice. VIP antagonist resulted in worsened disease of BALB/c mice. VIP proposed anti-inflammatory role in A. fumigatus keratitis.
| S-EPMC5824069 | biostudies-literature
Project description:Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5'-CAAGGT-3', in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.
| S-EPMC6940964 | biostudies-literature
Project description:The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ?dvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ?dvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ?dvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ?dvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ?dvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.
| S-EPMC2950423 | biostudies-literature
Project description:AIM:To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). METHODS:Murine models of A. fumigatus keratitis were established by scraping the central epithelium of mouse cornea, daubing A. fumigatus on the cornea and covering with a contact lens. The mice were randomly divided into the control group and the A. fumigatus-infected (A.F.) group for 1, 3 and 5d respectively, which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection. In this study, immunofluorescence staining was used to detect the expression and localization of AhR in mouse corneas, and the mRNA and protein of AhR were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. In addition, mouse peritoneal macrophages were stimulated by A. fumigatus with or without the pretreatment of AhR antagonist CH223191 and AhR agonist FICZ, and the tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and Arg-1 mRNA were detected by RT-PCR. RESULTS:According to the results of the slit light photography, it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3rd day after the infection of A. fumigatus. Contrasted with the control group, the expression of AhR in the corneal epithelial cells infected with A. fumigatus was significantly increased detected by immunofluorescence staining. AhR mainly expressed in the nucleus and cytoplasm of corneal epithelial cells. Consistent with the transcriptional level of AhR mRNA, the expression level of AhR protein reached the peak on the 3rd day after infection which was detected by Western blot. Furthermore, RT-PCR showed that CH223191 up-regulated the expression of TNF-α and iNOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages; inversely, FICZ reduced the expression of TNF-α and iNOS while elevated the expression of IL-10 and Arg-1. CONCLUSION:AhR is involved in the pathogenesis of A. fumigatus keratitis and induced immune protection in anti-A. fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.
| S-EPMC7013794 | biostudies-literature
Project description:Conidia of Aspergillus fumigatus adhere in vitro to host proteins and cells via the outer cell wall layer. The rodA gene of A. fumigatus was cloned by homology with the rodA gene of Aspergillus nidulans, which is involved in the structure of the rodlets characteristic of the surface layer. The A. fumigatus RODA protein sequence has 85% similarity to that of A. nidulans RODA; the sequence codes for a hydrophobin, a low-molecular-weight protein moderately hydrophobic and rich in cysteines. The gene was disrupted with the hygromycin B resistance gene. By transformation of protoplasts with the disrupted gene, RodA- mutants were generated. These mutants are deficient in the ability to disperse their conidia; their conidia lack the rodlet layer and are hydrophilic. The adhesion of the rodletless conidia to collagen and bovine serum albumin was lower than that of the wild type; in contrast, there was no difference between RodA- and RodA+ conidia in adhesion to pneumocytes, fibrinogen, and laminin, suggesting that RODA is not the receptor for these cells and proteins. RodA- conidia were pathogenic for mice.
| S-EPMC303120 | biostudies-other
Project description:Galactofuranose-containing molecules have been repeatedly shown to be important antigens among human fungal pathogens, including Aspergillus fumigatus. Immunogenic galactofuran determinants have been poorly characterized chemically, however. We reported here the characterization of two glycoproteins of A. fumigatus with an N-glycan containing galactofuranose. These proteins are a phospholipase C and a phytase. Chemical characterization of the N-glycan indicates that it is a mixture of Hex(5-13)HexNAc(2) oligosaccharides, the major molecular species corresponding to Hex(6-8)HexNAc(2). The N-glycan contained one galactofuranose unit that was in a terminal nonreducing position attached to the 2 position of Man. This single terminal nonreducing galactofuranose is essential for the immunoreactivity of the N-glycans assessed either with a monoclonal antibody that recognizes a tetra-beta-1,5-galactofuran chain of galactomannan or with Aspergillus-infected patient sera.
| S-EPMC1168957 | biostudies-literature
Project description:Upon infection of a host, the pathogenic fungus Aspergillus fumigatus is attacked by the reactive oxygen species produced by phagocytic cells. Detoxification of hydrogen peroxide by catalases was proposed as a way to overcome this host response. A. fumigatus produces three active catalases; one is produced by conidia, and two are produced by mycelia. The mycelial catalase Cat1p was studied previously. Here we characterized the two other catalases, their genes, and the phenotypes of gene-disrupted mutants. CatAp, a spore-specific monofunctional catalase, is resistant to heat, metal ions, and detergent. This enzyme is a dimeric protein with 84.5-kDa subunits. The 749-amino-acid polypeptide exhibits high levels of similarity to the Aspergillus nidulans CatA catalase and to bacterial catalase HPII of Escherichia coli. In spite of increased sensitivity to H(2)O(2), killing of DeltacatA conidia by alveolar macrophages and virulence in animals were similar to the killing of conidia by alveolar macrophages and virulence in animals observed for the wild type. In contrast to the Cat1p and CatAp catalases, the mycelial Cat2p enzyme is a bifunctional catalase-peroxidase and is sensitive to heat, metal ions, and detergent. This enzyme, an 82-kDa monomer, is homologous to catalase-peroxidases of several fungi and bacteria. Surprisingly, mycelium of the double Deltacat1Deltacat2 mutant with no catalase activity exhibited only slightly increased sensitivity to H(2)O(2) and was as sensitive to killing by polymorphonuclear neutrophils as mycelium of the wild-type strain. However, this mutant exhibited delayed infection in the rat model of aspergillosis compared to infection by the wild-type strain. These results indicate that conidial catalase is not a virulence factor and that mycelial catalases transiently protect the fungus from the host.
| S-EPMC155756 | biostudies-literature
Project description:BackgroundHydrophobins are a family of small secreted proteins with a characteristic pattern of eight cysteine residues found exclusively in filamentous fungi. They have originally been divided into two classes based on their physical properties and hydropathy patterns, and are involved in the attachment of hyphae to hydrophobic structures, the formation of aerial structures and appear to be involved in pathogenicity.FindingsAnalysis of nine genome sequences from seven Aspergilli revealed fifty hydrophobins, where each species displayed between two to eight hydrophobins. Twenty of the identified hydrophobins have not previously been described from these species. Apart from the cysteines, very little amino acid sequence homology was observed. Twenty-three of the identified hydrophobins could be classified as class I hydrophobins based on their conserved cysteine spacing pattern and hydropathy pattern. However twenty-six of the identified hydrophobins were intermediate forms. Notably, a single hydrophobin, ATEG_04730, from Aspergillus terreus displayed class II cysteine spacing and had a class II hydropathy pattern.ConclusionFifty hydrophobins were identified in Aspergillus, all containing the characteristic eight cysteine pattern. Aspergillus terreus exhibited both class I and class II hydrophobins. This is the first report of an Aspergillus species with the potential to express both class I and class II hydrophobins. Many of the identified hydrophobins could not directly be allocated to either class I or class II.
| S-EPMC3020181 | biostudies-literature