Expression of immune response genes in human corneal epithelial cells interacting with Asperrgillus flavus conidia
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PROVIDER: PRJNA733997 | ENA |
REPOSITORIES: ENA
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Project description:Human corneal epithelial cells internalize Aspergillus flavus conidia. But the biochemical response of corneal epithelial cells in response to A.flavus is not clear. In this study, NanoString analysis was used to study the immune genes expression in primary corneal epithelial cells and cell line infected with A. flavus conidia. The study revealed the differential expression of immune genes associated with TNF, Th17, NF-kB signaling and B cell receptor signaling in corneal epithelial cells. There is also difference in the response between cell line and primary cells to A. flavus implies the use of both cells to understand the host-pathogen interaction.
2021-06-01 | GSE175824 | GEO
Project description:Mycotic leratitis is the corneal inflammation predominantly caused by Fusarium and Aspergillus species. Corneal epithelium is the earliest cell type encounter the invading pathogen. The innate immune responses of human corneal epithelial cells against Aspergillus flavus is not known. Here we studied the role of human corneal epithelial cells against Aspergillus flavus infection. The results showed that corneal epithelial cells internalized Aspergillus flavus conidia through actin mediated polymerization surrounding the conidia. Further the actin inhibitor cytochalasin D treatment reduced the formation actin ring around the conidia. The engulfed conidia acquired endosomal proteins as revealed by immunofluorescence analsyisis. Mass spectromtery of phagosomal proteins confirmed the recruitment of endosomal proteins and other proteins involved in phagocytosis. These results show the involvement of corneal epithelial cells in anti fungal defense.
2021-04-07 | PXD019512 | Pride
Project description:BackgroundAspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics.MethodsHuman corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes.ResultsDifferent morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells.ConclusionsCombining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.
| S-EPMC8728928 | biostudies-literature
Project description:A. flavus exoproteome reference map as well as identification of the exoproteins from corneal isolates.
2015-01-07 | PXD001296 | Pride
Project description:Aspergillus flavus strain:corneal isolates Genome sequencing and assembly
| PRJNA445644 | ENA
Project description:Dr. Panjwani's laboratory is focusing on the mechanism by which galectins-3 and 7 mediate corneal epithelial cell migration. We are currently performing studies to: (i) identify and characterize the corneal epithelial cell surface and extracellular matrix (ECM) molecules which serve as counterreceptors of galectin-3 and -7, to establish whether the lectins modulate corneal epithelial cell migration by binding to well known integrins, growth factor receptors, and/or ECM molecules and (ii) determine whether galectin-3 mediates corneal epithelial cell migration indirectly by modulating the expression of key adhesion and/or signal transduction molecules by using small interfering RNA, cDNA microarrays and glycogene arrays. We have prepared three independent preparations of total RNA of corneal epithelial cells from WT mice (total six samples) for analysis of glycogene expression. Samples are Normal Cornea (Left eye) and Laser ablation + 16-18 hours healing (right eye)
2010-12-11 | E-GEOD-26009 | biostudies-arrayexpress
Project description:Objective: Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of GO analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. This study aimed to investigate the integrative function of the aflR gene in A. flavus. Design: In this study, we used Aspergillus flavus NRRL3357 as a wild-type strain (WT) and constructed a knockout strain of A. flavus ΔaflR by homologous recombination. Based on the transcriptomics technology, we investigated the metabolic effects of aflR gene on growth, development and toxin synthesis of A. flavus, and discussed the overall regulation mechanism of aflR gene on A. flavus at the transcriptional level. Results: The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. In addition, disrupted strains of the aflR gene produced relatively sparse conidia and a very small number of sclerotia. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly down-regulated. Meanwhile, the ΔaflR strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT strain. Conclusions: The results showed that the A. flavus aflR gene also played a positive role in the growth and development of fungi.
2021-07-16 | GSE179978 | GEO
Project description:Linking cell reproduction and survival is a key task of all life forms. All fungi in the genus Aspergillus reproduce by forming asexual spores called conidia, of which formation is governed by the central regulatory circuit, BrlA->AbaA->WetA. Here, we report that WetA is a key multi-functional regulator that bridged spore differentiation, long-term survival, and chemical development in Aspergillus flavus.
2017-06-04 | GSE95711 | GEO
Project description:Aspergillus flavus and A. parasiticus are two of the most important aflatoxin-producing species that contaminate agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here, we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four out of forty-five attempted interspecific crosses between compatible mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were isolated and were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of putative allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important species.
2015-10-01 | GSE61981 | GEO
Project description:A. flavus keratitis is one of the predominant fungal infections affecting the tropical parts of the world. Melanin is a virulence factor in numerous pathogenic fungi and the melanin layer maintains the cell wall structure and provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile and virulence of melanized and non-melanized conidia of A. flavus. An environmental isolate and two clinical isolates were included in this study. L-DOPA melanin pathway-specific inhibitor kojic acid (KA) as used to prepare non-melanized conidia. Conidial surface proteins were extracted using 100% formic acid at 0 ºC and the extracted proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Formic acid extracts were analyzed in an Orbitrap Velos pro-mass spectrometer. Conidial surface morphology difference was examined using scanning electron microscopy (SEM). Furthermore, lactophenol and calcofluor staining were performed to check the morphology and staining efficiency of melanized conidia (MC) and non-melanized conidia (NMC). A Galleria mellonella insect model was used to examine the virulence efficiency of MC and NMC. Kojic acid treatment inhibited melanin synthesis in A. flavus and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathway were found only in the formic acid extracts of NMC. SEM analysis showed the conidial surface morphology difference between the NMC and MC indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining of MC and NMC. Evaluation of virulence of MC and NMC in G. mellonella model showed NMC was less virulent compared to MC . Our results showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer and hence melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. G. mellonella in vivo virulence assay also confirmed that the non-melanized conidia were susceptible to host defense as in other Aspergillus pathogens.
2024-05-23 | PXD037707 | Pride