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Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert.

ABSTRACT: BACKGROUND:Aspergillus fumigatus is the main causative agent of aspergillosis. Infections rarely occur in immunocompetent individuals, indicating efficient clearance of conidia by pulmonary defense mechanisms. Other aspergilli like Aspergillus niger also cause infections but to a much lesser extent. Our previous studies showed that A. fumigatus and A. niger have different behavior in the presence of type II alveolar A549 epithelial cells. A. fumigatus conidia are more efficiently internalized by these cells and germination is delayed when compared to A. niger. In addition, hyphae that have escaped the epithelial cells grow parallel to the epithelium, while A. niger grows away from this cell layer. RESULTS:Here it is shown that global gene expression of A. fumigatus and A. niger is markedly different upon contact with A549 cells. A total of 545 and 473 genes of A. fumigatus and A. niger, respectively, were differentially expressed when compared to growth in the absence of A549 cells. Notably, only 53 genes (approximately 10%) were shared in these gene sets. The different response was also illustrated by the fact that only 4 out of 75 GO terms were shared that were enriched in the differentially expressed gene sets. The orthologues of A. fumigatus genes involved in hypoxia regulation and heat shock were also up-regulated in A. niger, whereas thioredoxin reductase and allergen genes were found up-regulated in A. fumigatus but down-regulated in A. niger. Infection with A. fumigatus resulted in only 62 up and 47 down-regulated genes in A549. These numbers were 17 and 34 in the case of A. niger. GO terms related with immune response were down-regulated upon exposure to A. fumigatus but not in the case of A. niger. This indicates that A. fumigatus reprograms A549 to be less immunologically alert. CONCLUSIONS:Our dual transcriptomic analysis supports earlier observations of a marked difference in life style between A. fumigatus and A. niger when grown in the presence of type II epithelial cells. The results indicate important differences in gene expression, amongst others down regulation of immune response genes in lung epithelial cells by A. fumigatus but not by A niger.

SUBMITTER: Escobar N

PROVIDER: S-EPMC6044037 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert.

Escobar Natalia N Valdes Ivan D ID Keizer Esther M EM Ordonez Soledad R SR Ohm Robin A RA Wösten Han A B HAB de Cock Hans H

BMC genomics 20180713 1

<h4>Background</h4>Aspergillus fumigatus is the main causative agent of aspergillosis. Infections rarely occur in immunocompetent individuals, indicating efficient clearance of conidia by pulmonary defense mechanisms. Other aspergilli like Aspergillus niger also cause infections but to a much lesser extent. Our previous studies showed that A. fumigatus and A. niger have different behavior in the presence of type II alveolar A549 epithelial cells. A. fumigatus conidia are more efficiently interna ...[more]

PMID: 30005605

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Project description:For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

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Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert.

Publications

Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert.

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