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Dual organism transcriptomics of airway epithelial cells interacting with conidia of Aspergillus fumigatus.

Oosthuizen JL, Gomez P, Ruan J, Hackett TL, Moore MM, Knight DA, Tebbutt SJ - PLoS ONE (2011)

Bottom Line: Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity.The up-regulation of IL-6 by epithelia and simultaneous activation of several pathways by fungal conidia warrants further investigation as we seek to better understand this interaction in both health and disease.The cellular response of the airway epithelium to A. fumigatus is important to understand if we are to improve host-pathogen outcomes.

View Article: PubMed Central - PubMed

Affiliation: UBC James Hogg Research Centre, Institute for HEART+LUNG Health, Providence Health Care, Vancouver, British Columbia, Canada.

ABSTRACT

Background: Given the complex nature of the responses that can occur in host-pathogen interactions, dual transcriptomics offers a powerful method of elucidating these interactions during infection. The gene expression patterns of Aspergillus fumigatus conidia or host cells have been reported in a number of previous studies, but each focused on only one of the interacting organisms. In the present study, we profiled simultaneously the transcriptional response of both A. fumigatus and human airway epithelial cells (AECs).

Methodology: 16HBE14o- transformed bronchial epithelial cells were incubated with A. fumigatus conidia at 37°C for 6 hours, followed by genome-wide transcriptome analysis using human and fungal microarrays. Differentially expressed gene lists were generated from the microarrays, from which biologically relevant themes were identified. Human and fungal candidate genes were selected for validation, using RT-qPCR, in both 16HBE14o- cells and primary AECs co-cultured with conidia.

Principal findings: We report that ontologies related to the innate immune response are activated by co-incubation with A. fumigatus condia, and interleukin-6 (IL-6) was confirmed to be up-regulated in primary AECs via RT-qPCR. Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity.

Conclusion: To our knowledge, this is the first study to apply a dual organism transcriptomics approach to interactions of A. fumigatus conidia and human airway epithelial cells. The up-regulation of IL-6 by epithelia and simultaneous activation of several pathways by fungal conidia warrants further investigation as we seek to better understand this interaction in both health and disease. The cellular response of the airway epithelium to A. fumigatus is important to understand if we are to improve host-pathogen outcomes.

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Relative mRNA expression levels of A. fumigatus genes as determined by RT-qPCR.RNA was obtained from four co-incubations each of A. fumigatus conidia with 16HBE14o- (grey bars) or primary human bronchial epithelial cells (AECs) (white bars). Height of each bar represents expression of gene in co-incubated condition relative to conida alone control (mean ± SE). (* p<0.05).
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pone-0020527-g003: Relative mRNA expression levels of A. fumigatus genes as determined by RT-qPCR.RNA was obtained from four co-incubations each of A. fumigatus conidia with 16HBE14o- (grey bars) or primary human bronchial epithelial cells (AECs) (white bars). Height of each bar represents expression of gene in co-incubated condition relative to conida alone control (mean ± SE). (* p<0.05).

Mentions: Among the most prominent annotated functions identified involved genes classified in vacuolar acidification and metallopeptidase activities. To validate the expression levels generated by the microarray, eight genes in total were chosen for further analysis with RT-qPCR (Table 2). Four of these genes, metallopeptidase MepB, matrix AAA protease MAP-1, sulphur metabolism regulator SkpA, and the vacuolar ATPase 98 kDa subunit, were chosen as gene targets of interest based on the significance of their GOEAST classifications. MepB and Map-1 are part of the metallopeptidase classification, while SkpA and the Vacuolar ATPase 98 kDa subunit are associated with the vacuolar acidification gene set. The selection of the other four targets, tubulin-specific chaperone C, NAD-dependent formate dehydrogenase (fdh), β-glucosidase, and L-ornithine N5-oxygenase (SidA), was based on significance at the single gene level. We have an interest in iron metabolism by A. fumigatus, and siderophore-mediated iron acquisition is critical for virulence [24], [25]. These genes were tested in two different incubation types, one involving 16HBE14o- cells and the other a co-incubation with AECs (Figure 3). Interestingly, all genes achieved significance following incubation with at least one cell type, with the exception of MepB. The vacuolar ATPase 98 kDA subunit, SkpA, and MAP-1 were significantly differentially expressed following incubations with both 16HBE14o- and AECs. Significance was achieved only in 16HBE14o- cells for fdh and SidA, whereas the tubulin-specific chaperone C and β-glucosidase were significant only in the AECs.


Dual organism transcriptomics of airway epithelial cells interacting with conidia of Aspergillus fumigatus.

Oosthuizen JL, Gomez P, Ruan J, Hackett TL, Moore MM, Knight DA, Tebbutt SJ - PLoS ONE (2011)

Relative mRNA expression levels of A. fumigatus genes as determined by RT-qPCR.RNA was obtained from four co-incubations each of A. fumigatus conidia with 16HBE14o- (grey bars) or primary human bronchial epithelial cells (AECs) (white bars). Height of each bar represents expression of gene in co-incubated condition relative to conida alone control (mean ± SE). (* p<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105077&req=5

pone-0020527-g003: Relative mRNA expression levels of A. fumigatus genes as determined by RT-qPCR.RNA was obtained from four co-incubations each of A. fumigatus conidia with 16HBE14o- (grey bars) or primary human bronchial epithelial cells (AECs) (white bars). Height of each bar represents expression of gene in co-incubated condition relative to conida alone control (mean ± SE). (* p<0.05).
Mentions: Among the most prominent annotated functions identified involved genes classified in vacuolar acidification and metallopeptidase activities. To validate the expression levels generated by the microarray, eight genes in total were chosen for further analysis with RT-qPCR (Table 2). Four of these genes, metallopeptidase MepB, matrix AAA protease MAP-1, sulphur metabolism regulator SkpA, and the vacuolar ATPase 98 kDa subunit, were chosen as gene targets of interest based on the significance of their GOEAST classifications. MepB and Map-1 are part of the metallopeptidase classification, while SkpA and the Vacuolar ATPase 98 kDa subunit are associated with the vacuolar acidification gene set. The selection of the other four targets, tubulin-specific chaperone C, NAD-dependent formate dehydrogenase (fdh), β-glucosidase, and L-ornithine N5-oxygenase (SidA), was based on significance at the single gene level. We have an interest in iron metabolism by A. fumigatus, and siderophore-mediated iron acquisition is critical for virulence [24], [25]. These genes were tested in two different incubation types, one involving 16HBE14o- cells and the other a co-incubation with AECs (Figure 3). Interestingly, all genes achieved significance following incubation with at least one cell type, with the exception of MepB. The vacuolar ATPase 98 kDA subunit, SkpA, and MAP-1 were significantly differentially expressed following incubations with both 16HBE14o- and AECs. Significance was achieved only in 16HBE14o- cells for fdh and SidA, whereas the tubulin-specific chaperone C and β-glucosidase were significant only in the AECs.

Bottom Line: Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity.The up-regulation of IL-6 by epithelia and simultaneous activation of several pathways by fungal conidia warrants further investigation as we seek to better understand this interaction in both health and disease.The cellular response of the airway epithelium to A. fumigatus is important to understand if we are to improve host-pathogen outcomes.

View Article: PubMed Central - PubMed

Affiliation: UBC James Hogg Research Centre, Institute for HEART+LUNG Health, Providence Health Care, Vancouver, British Columbia, Canada.

ABSTRACT

Background: Given the complex nature of the responses that can occur in host-pathogen interactions, dual transcriptomics offers a powerful method of elucidating these interactions during infection. The gene expression patterns of Aspergillus fumigatus conidia or host cells have been reported in a number of previous studies, but each focused on only one of the interacting organisms. In the present study, we profiled simultaneously the transcriptional response of both A. fumigatus and human airway epithelial cells (AECs).

Methodology: 16HBE14o- transformed bronchial epithelial cells were incubated with A. fumigatus conidia at 37°C for 6 hours, followed by genome-wide transcriptome analysis using human and fungal microarrays. Differentially expressed gene lists were generated from the microarrays, from which biologically relevant themes were identified. Human and fungal candidate genes were selected for validation, using RT-qPCR, in both 16HBE14o- cells and primary AECs co-cultured with conidia.

Principal findings: We report that ontologies related to the innate immune response are activated by co-incubation with A. fumigatus condia, and interleukin-6 (IL-6) was confirmed to be up-regulated in primary AECs via RT-qPCR. Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity.

Conclusion: To our knowledge, this is the first study to apply a dual organism transcriptomics approach to interactions of A. fumigatus conidia and human airway epithelial cells. The up-regulation of IL-6 by epithelia and simultaneous activation of several pathways by fungal conidia warrants further investigation as we seek to better understand this interaction in both health and disease. The cellular response of the airway epithelium to A. fumigatus is important to understand if we are to improve host-pathogen outcomes.

Show MeSH
Related in: MedlinePlus