Human cells infected with Mucormycosis-causing strains from clinical settings

Mucormycosis is an increasingly common, life-threatening fungal infection caused by fungi belonging to the subphylum Mucormycotina, order Mucorales. The major risk factors for mucormycosis include uncontrolled diabetes mellitus, treatment with corticosteroids, organ or bone marrow transplantation, neutropenia, trauma and burns, malignant hematological disorders, and deferoxamine-therapy in patients receiving hemodialysis. Amphotericin B (AmB) remains the only antifungal agent approved for the treatment of invasive mucormycosis. Because fungi belonging to the mucorales order are relatively resistant to AmB, high doses are required, which frequently causes nephrotoxicity and other adverse effects. In the absence of surgical removal of the infected focus (such as excision of the eye in patients with rhinocerebral mucormycosis), antifungal therapy alone is rarely curative. Even when surgical debridement is combined with high-dose AmB, the mortality associated with mucor¬mycosis is >50%. In patients with prolonged neutropenia or disseminated disease, mortality is 90-100%. Rhizopus oryzae is by far the most common organism isolated from patients with mucormycosis, and is responsible for approximately 70% of all cases of mucormycosis. Recent epidemiological data collected from transplant centers have demonstrated a striking increase in the number of reported cases of mucormycosis. Furthermore, mucormycosis outbreaks are also known to follow natural disasters as was the case following the tsunami that devastated Indonesia in 2004 and the tornadoes that destroyed Joplin, Missouri in June 2011. Hence, there exists a large impetus to identify particular virulence factors and biomarkers associated with mucormycosis, which would lead to a rational approach to develop new antifungals and vaccines to prevent deaths specifically due to mucormycosis. In order to better understand the complex nature of the host pathogen interaction during mucormycosis, we performed dual species RNA-seq on 5 different Mucorales strains during in vitro infection of endothelial or epithelial cells. 5 different strains of Mucorales (see below) were used to infect two different types of human tissue culture monolayers of either epithelial cells or endothelial cells. RNA from the infection mixtures were harvested at 90 minutes post infection. Sample name|title|source name|infection|organism|cell type|molecule|description|processed data file |SRA accession Absidia corymbifera 008-049 + endothelial cells|A1|Absidia corymbifera in Endothelial cells|Absidia corymbifera 008-049|human|Endothelial cells|total RNA|Absidia corymbifera in Endothelial cells|A1.genic.coverage.stats.rpkm.txt|SRX365460 Absidia corymbifera 008-049 + HUVEC media alone|A2|Absidia corymbifera in HUVEC media|Absidia corymbifera 008-049|human|HUVEC media|total RNA|Absidia corymbifera in HUVEC media||SRX365522 Absidia corymbifera 008-049 + epithelial cells|A3|Absidia corymbifera in epithelial cells|Absidia corymbifera 008-049|human|epithelial cells|total RNA|Absidia corymbifera in epithelial cells|A3.genic.coverage.stats.rpkm.txt|SRX365523 Absidia corymbifera008-049 + epithelial media alone|A4|Absidia corymbifera in epithelial cell media|Absidia corymbifera 008-049|human|epithelial cell media|total RNA|Absidia corymbifera in epithelial cell media||SRX365541 Cunninghamella bertholttiae 175 + endothelial cells|C1|Cunninghamella bertholttiae in Endothelial cells|Cunninghamella bertholttiae 175|human|Endothelial cells|total RNA|Cunninghamella bertholttiae in Endothelial cells|C1.genic.coverage.stats.rpkm.txt|SRX365542 Cunninghamella bertholttiae 175 + HUVEC media alone|C2|Cunninghamella bertholttiae in HUVEC media|Cunninghamella bertholttiae 175|human|HUVEC media|total RNA|Cunninghamella bertholttiae in HUVEC media||SRX365619 Cunninghamella bertholttiae 175 + epithelial cells|C3|Cunninghamella bertholttiae in epithelial cells|Cunninghamella bertholttiae 175|human|epithelial cells|total RNA|Cunninghamella bertholttiae in epithelial cells|C3.genic.coverage.stats.rpkm.txt|SRX365620 Cunninghamella bertholttiae 175 + epithelial media alone|C4|Cunninghamella bertholttiae in epithelial cell media|Cunninghamella bertholttiae 175|human|epithelial cell media|total RNA|Cunninghamella bertholttiae in epithelial cell media||SRX365623 Mucor ramosissumus 97-1192 + endothelial cells|M1|Mucor ramosissumus in Endothelial cells|Mucor ramosissumus 97-1192|human|Endothelial cells|total RNA|Mucor ramosissumus in Endothelial cells| M1.genic.coverage.stats.rpkm.txt|SRX365603 Mucor ramosissumus 97-1192 + HUVEC media alone|M2|Mucor ramosissumus in HUVEC media|Mucor ramosissumus 97-1192|human|HUVEC media|total RNA|Mucor ramosissumus in HUVEC media||SRX365655 Mucor ramosissumus 97-1192 + epithelial cells|M3|Mucor ramosissumus in epithelial cells|Mucor ramosissumus 97-1192|human|epithelial cells|total RNA|Mucor ramosissumus in epithelial cells| M3.genic.coverage.stats.rpkm.txt|SRX365662 Mucor ramosissumus 97-1192 + epithelial media alone|M4|Mucor ramosissumus in epithelial cell media|Mucor ramosissumus 97-1192|human|epithelial cell media|total RNA|Mucor ramosissumus in epithelial cell media||SRX365664 Rhizopus oryzea 99-880 + endothelial cells|880_1|Rhizopus oryzea in Endothelial cells|Rhizopus oryzea 99-880|human|Endothelial cells|total RNA|Rhizopus oryzea in Endothelial cells|880_1.genic.coverage.stats.rpkm.txt|SRX365613 Rhizopus oryzea 99-880 + HUVEC media alone|880_2|Rhizopus oryzea in HUVEC media|Rhizopus oryzea 99-880|human|HUVEC media|total RNA|Rhizopus oryzea in HUVEC media||SRX365624 Rhizopus oryzea 99-880 + epithelial cells|880_3|Rhizopus oryzea in epithelial cells|Rhizopus oryzea 99-880|human|epithelial cells|total RNA|Rhizopus oryzea in epithelial cells|880_3.genic.coverage.stats.rpkm.txt|SRX365628 Rhizopus oryzea 99-880 + epithelial media alone|880_4|Rhizopus oryzea in epithelial cell media|Rhizopus oryzea 99-880|human|epithelial cell media|total RNA|Rhizopus oryzea in epithelial cell media||SRX365629 Rhizopus oryzea 99-892 + endothelial cells|892_1|Rhizopus oryzea in Endothelial cells|Rhizopus oryzea 99-892|human|Endothelial cells|total RNA|Rhizopus oryzea in Endothelial cells|892_1.genic.coverage.stats.rpkm.txt|SRX365618 Rhizopus oryzea 99-892 + HUVEC media alone|892_2|Rhizopus oryzea in HUVEC media|Rhizopus oryzea 99-892|human|HUVEC media|total RNA|Rhizopus oryzea in HUVEC media||SRX365656 Rhizopus oryzea 99-892 + epithelial cells|892_3|Rhizopus oryzea in epithelial cells|Rhizopus oryzea 99-892|human|epithelial cells|total RNA|Rhizopus oryzea in epithelial cells|892_3.genic.coverage.stats.rpkm.txt|SRX365657 Rhizopus oryzea 99-892 + epithelial media alone|892_4|Rhizopus oryzea in epithelial cell media|Rhizopus oryzea 99-892|human|epithelial cell media|total RNA|Rhizopus oryzea in epithelial cell media||SRX365658 Cells from each of the Mucorales strains were used to infect endothelial cells or epithelial cells in tissue culture media for 90 minutes at 37 degrees celcius. The negative control samples were the host or C. albicans cells alone in tissue culture media for the same amount of time. RNA extractions were performed using RiboPure RNA Purification Kit, yeast (Life Technologies, AM1926) following manufacturer's protocols RNA libraries were prepared for sequencing using standard Illumina protocols RNA-Seq Base calls were performed using the bulit in Illumina basecaller. Sequenced reads were mapped to each fungal genome and the human genome using TopHat v. 1.4. RPKM values were calculated for the genes in the human genome. Genome Build: Homo sapiens.GRCh37.71