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Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions

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Abstract
Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.
Keywords
GENE-EXPRESSION, RNA, INFECTION, CANDIDIASIS, DNA

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Citation

Please use this url to cite or link to this publication:

MLA
Rodriguez Sánchez, Antonio, et al. “Nucleic Acids Enrichment of Fungal Pathogens to Study Host-Pathogen Interactions.” SCIENTIFIC REPORTS, vol. 9, 2019.
APA
Rodriguez Sánchez, A., Guillemyn, B., Coucke, P., & Vaneechoutte, M. (2019). Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions. SCIENTIFIC REPORTS, 9.
Chicago author-date
Rodriguez Sánchez, Antonio, Brecht Guillemyn, Paul Coucke, and Mario Vaneechoutte. 2019. “Nucleic Acids Enrichment of Fungal Pathogens to Study Host-Pathogen Interactions.” SCIENTIFIC REPORTS 9.
Chicago author-date (all authors)
Rodriguez Sánchez, Antonio, Brecht Guillemyn, Paul Coucke, and Mario Vaneechoutte. 2019. “Nucleic Acids Enrichment of Fungal Pathogens to Study Host-Pathogen Interactions.” SCIENTIFIC REPORTS 9.
Vancouver
1.
Rodriguez Sánchez A, Guillemyn B, Coucke P, Vaneechoutte M. Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions. SCIENTIFIC REPORTS. 2019;9.
IEEE
[1]
A. Rodriguez Sánchez, B. Guillemyn, P. Coucke, and M. Vaneechoutte, “Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions,” SCIENTIFIC REPORTS, vol. 9, 2019.
@article{8640808,
  abstract     = {Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.},
  articleno    = {18037},
  author       = {Rodriguez Sánchez, Antonio and Guillemyn, Brecht and Coucke, Paul and Vaneechoutte, Mario},
  issn         = {2045-2322},
  journal      = {SCIENTIFIC REPORTS},
  keywords     = {GENE-EXPRESSION,RNA,INFECTION,CANDIDIASIS,DNA},
  language     = {eng},
  pages        = {9},
  title        = {Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions},
  url          = {http://dx.doi.org/10.1038/s41598-019-54608-x},
  volume       = {9},
  year         = {2019},
}

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