scholarly journals The Gβ-like Protein AfCpcB Affects Sexual Development, Response to Oxidative Stress and Phagocytosis by Alveolar Macrophages in Aspergillus fumigatus

2022 ◽  
Vol 8 (1) ◽  
pp. 56
Author(s):  
Joo-Yeon Lim ◽  
Yeon-Ju Kim ◽  
Hee-Moon Park
Keyword(s):  
Oxidative Stress ◽  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Alveolar Macrophages ◽  
Sexual Development ◽  
Reproductive Organ ◽  
Transcriptional Analysis ◽  
Ergosterol Biosynthesis ◽  
Protein Signaling

G-protein signaling is important for signal transduction, allowing various stimuli that are external to a cell to affect its internal molecules. In Aspergillus fumigatus, the roles of Gβ-like protein CpcB on growth, asexual development, drug sensitivity, and virulence in a mouse model have been previously reported. To gain a deeper insight into Aspergillus fumigatus sexual development, the ΔAfcpcB strain was generated using the supermater AFB62 strain and crossed with AFIR928. This cross yields a decreased number of cleistothecia, including few ascospores. The sexual reproductive organ-specific transcriptional analysis using RNAs from the cleistothecia (sexual fruiting bodies) indicated that the CpcB is essential for the completion of sexual development by regulating the transcription of sexual genes, such as veA, steA, and vosA. The ΔAfcpcB strain revealed increased resistance to oxidative stress by regulating genes for catalase, peroxiredoxin, and ergosterol biosynthesis. The ΔAfcpcB strain showed decreased uptake by alveolar macrophages in vitro, decreased sensitivity to Congo red, decreased expression of cell wall genes, and increased expression of the hydrophobin genes. Taken together, these findings indicate that AfCpcB plays important roles in sexual development, phagocytosis by alveolar macrophages, biosynthesis of the cell wall, and oxidative stress response.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

scholarly journals Species-Specific Immunological Reactivities Depend on the Cell-Wall Organization of the Two Aspergillus, Aspergillus fumigatus and A. flavus

2021 ◽  
Vol 11 ◽  
Author(s):  
Sarah Sze Wah Wong ◽  
Lakshmi Prabha Venugopalan ◽  
Audrey Beaussart ◽  
Anupama Karnam ◽  
Mohammed Razeeth Shait Mohammed ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Inflammatory Responses ◽  
Treatment Strategies ◽  
Specific Treatment ◽  
Antifungal Drugs ◽  
Cell Wall Polysaccharides ◽  
Superficial Infection ◽  
Species Specific

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.

scholarly journals Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

2020 ◽  
Author(s):  
Isabel Karkossa ◽  
Anne Bannuscher ◽  
Bryan Hellack ◽  
Wendel Wohlleben ◽  
Julie Laloy ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Alveolar Macrophages ◽  
Model System ◽  
Alveolar Epithelial Cells ◽  
Model Systems ◽  
Alveolar Epithelial ◽  
Different Levels

Abstract Background The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, alveolar epithelial cells and alveolar macrophages were treated with different doses of NMs, and the results were compared to effects on rat lungs after short-term inhalations and instillations at varying doses with and without a recovery period.Results Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses confirmed oxidative stress as a significant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to inflammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo.Conclusions The consistent classification indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

scholarly journals Human IgM Inhibits the Formation of Titan-Like Cells in Cryptococcus neoformans

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Nuria Trevijano-Contador ◽  
Kaila M. Pianalto ◽  
Connie B. Nichols ◽  
Oscar Zaragoza ◽  
J. Andrew Alspaugh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stress Response ◽  
B Cell ◽  
Cryptococcus Neoformans ◽  
Cell Formation ◽  
Transcriptional Analysis ◽  
Content Type ◽  
Human Immunoglobulins ◽  
Genes Encoding

ABSTRACT Human studies have shown associations between cryptococcal meningitis and reduced IgM memory B cell levels, and studies in IgM- and/or B cell-deficient mice have demonstrated increased Cryptococcus neoformans dissemination from lungs to brain. Since immunoglobulins are part of the immune milieu that C. neoformans confronts in a human host, and its ability to form titan cells is an important virulence mechanism, we determined the effect of human immunoglobulins on C. neoformans titan cell formation in vitro. (i) Fluorescence microscopy showed normal human IgG and IgM bind C. neoformans. (ii) C. neoformans grown in titan cell-inducing medium with IgM, not IgG, inhibited titan-like cell formation. (iii) Absorption of IgM with laminarin or curdlan (branched and linear 1-3-beta-d-glucans, respectively) decreased this effect. (iv) Transmission electron microscopy revealed that cells grown with IgM had small capsules and unique features not seen with cells grown with IgG. (v) Comparative transcriptional analysis of cell wall, capsule, and stress response genes showed that C. neoformans grown with IgM, not IgG or phosphate-buffered saline (PBS), had decreased expression of chitin synthetase, CHS1, CHS2, and CHS8, and genes encoding cell wall carbohydrate synthetases α-1-3-glucan (AGS1) and β-1,3-glucan (FKS1). IgM also decreased expression of RIM101 and HOG1, genes encoding central regulators of C. neoformans stress response pathways and cell morphogenesis. Our data show human IgM affects C. neoformans morphology in vitro and suggest that the hypothesis that human immunoglobulins may affect C. neoformans virulence in vivo warrants further investigation.

scholarly journals O-Mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) Is Crucial for Cell Wall Integrity and Conidium Morphology, Especially at an Elevated Temperature

2007 ◽  
Vol 6 (12) ◽  
pp. 2260-2268 ◽  
Author(s):  
Hui Zhou ◽  
Hongyan Hu ◽  
Lijuan Zhang ◽  
Ruoyu Li ◽  
Haomiao Ouyang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Elevated Temperature ◽  
Aspergillus Fumigatus ◽  
Cell Wall Integrity ◽  
Secretory Proteins ◽  
Experimental Conditions ◽  
Human Fungal Pathogen ◽  
Conidium Formation

ABSTRACT Protein O-mannosyltransferases initiate O mannosylation of secretory proteins, which are of fundamental importance in eukaryotes. In this study, the PMT gene family of the human fungal pathogen Aspergillus fumigatus was identified and characterized. Unlike the case in Saccharomyces cerevisiae, where the PMT family is highly redundant, only one member of each PMT subfamily, namely, Afpmt1, Afpmt2, and Afpmt4, is present in A. fumigatus. Mutants with a deletion of Afpmt1 are viable. In vitro and in vivo activity assays confirmed that the protein encoded by Afpmt1 acts as an O-mannosyltransferase (AfPmt1p). Characterization of the ΔAfpmt1 mutant showed that a lack of AfPmt1p results in sensitivity to elevated temperature and defects in growth and cell wall integrity, thereby affecting cell morphology, conidium formation, and germination. In a mouse model, Afpmt1 was not required for the virulence of A. fumigatus under the experimental conditions used.

scholarly journals Synergy, Pharmacodynamics, and Time-Sequenced Ultrastructural Changes of the Interaction between Nikkomycin Z and the Echinocandin FK463 against Aspergillus fumigatus

2001 ◽  
Vol 45 (12) ◽  
pp. 3310-3321 ◽  
Author(s):  
Christine C. Chiou ◽  
Nikolaos Mavrogiorgos ◽  
Elizabeth Tillem ◽  
Richard Hector ◽  
Thomas J. Walsh
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Rhizopus Oryzae ◽  
Ultrastructural Changes ◽  
Synergistic Activity ◽  
Partial Recovery ◽  
Electron Microscopic ◽  
Wide Range ◽  
Nikkomycin Z

ABSTRACT We investigated the potential synergy between two cell wall-active agents, the echinocandin FK463 (FK) and the chitin synthase inhibitor nikkomycin Z (NZ), against 16 isolates of filamentous fungi. Susceptibility testing was performed with a broth macrodilution procedure by NCCLS methods. The median minimal effective concentration (MEC) of FK against all Aspergillus species was 0.25 μg/ml (range, 0.05 to 0.5 μg/ml). For Fusarium solaniand Rhizopus oryzae, MECs of FK were >512 μg/ml. The median MEC of NZ against Aspergillus fumigatus was 32 μg/ml (range, 8 to 64 μg/ml), and that against R. oryzae was 0.5 μg/ml (range, 0.06 to 2 μg/ml); however, for the other Aspergillus species, as well as F. solani, MECs were >512 μg/ml. A checkerboard inhibitory assay demonstrated synergy against A. fumigatus (median fractional inhibitory concentration index = 0.312 [range, 0.15 to 0.475]). The effect was additive to indifferent against R. oryzae and indifferent against other Aspergillus spp. and F. solani. We further investigated the pharmacodynamics of hyphal damage by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and examined the time-sequenced changes in hyphal ultrastructure. Significant synergistic hyphal damage was demonstrated with the combination of NZ (2 to 32 μg/ml) and FK (0.03 to 0.5 μg/ml) over a wide range of concentrations (P < 0.001). The synergistic effect was most pronounced after 12 h of incubation and was sustained through 24 h. Time-sequenced light and electron microscopic studies demonstrated that structural alterations of hyphae were profound, with marked transformation of hyphae to blastospore-like structures, in the presence of FK plus NZ, while fungi treated with a single drug showed partial recovery at 24 h. The methods used in this study may be applicable to elucidating the activity and interaction of other cell wall-active agents. In summary, these two cell wall-targeted antifungal agents, FK and NZ, showed marked time-dependent in vitro synergistic activity against A. fumigatus.

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

2005 ◽  
Vol 12 (9) ◽  
pp. 1063-1068 ◽  
Author(s):  
Ashok K. Chaturvedi ◽  
A. Kavishwar ◽  
G. B. Shiva Keshava ◽  
P. K. Shukla
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Protective Efficacy ◽  
Wide Spectrum ◽  
Kidney Tissue ◽  
Survival Times ◽  
Fungal Cell ◽  
New Strategy

ABSTRACT 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.

scholarly journals Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

2015 ◽  
Vol 59 (10) ◽  
pp. 5932-5941 ◽  
Author(s):  
Louise A. Walker ◽  
Keunsook K. Lee ◽  
Carol A. Munro ◽  
Neil A. R. Gow
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Dynamic Imaging ◽  
Chitin Synthase ◽  
Response To Treatment ◽  
Chitin Synthesis ◽  
Calcofluor White ◽  
Content Type ◽  
Chitin Content

ABSTRACTTreatment ofAspergillus fumigatuswith echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca2+-calcineurin signaling pathways.A. fumigatusmutants with thechsgene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsGmutant was hypersensitive to caspofungin, and all other ΔAfchsmutants tested remained capable of increasing their chitin content in response to treatment with CaCl2and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchsmutants tested, with the exception of the ΔAfchsGmutant, which remained sensitive to caspofungin.In vitroexposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was againAfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. Thesein vitrodata demonstrate thatA. fumigatushas the potential to survive echinocandin treatmentin vivobyAfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

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