GSE114628
Dual RNAseq of murine alveolar macrophage cell line MH-S confronted with resting spores of Lichtheimia corymbifera JMRC:SF:09682
During the infection process, both the host and the pathogen undergo a dynamic cascade of events that result in altered gene expression patterns (Westermann et al. 2012). With dual-RNA analysis, the complex interaction between a pathogen and its host during infection can be revealed simultaneously at their RNA levels (Westermann et al. 2012). Therefore, dual-transcriptomic analysis on JMRC:FSU:09682 and MH-S was performed to understand the pathogenesis at the genetic level to identify what kind of stresses that L. corymbifera encounter as well as the transcriptional response of macrophages during the infection. L. corymbifera (JRMC:FSU:09682) was cultivated for 7 days in KK1 medium (Kraibooj et al. 2014) at 37 °C . Murine alveolar MH-S macrophages (ATCC: CRL-2019) were cultivated in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum at 37 °C in 5 % CO2. Macrophages were seeded in 6 well plates at 106 cells per well to adhere overnight. Macrophages were infected with spores at a MOI (Multiplicity of Infection) of 5. After 3 hr of co-incubation with MH-S, extracellular spores were removed by at least 3 washing steps with pre-warmed RPMI-1640 (PAA Laboratories) and incubated for 13 hr in prior to the RNA extraction procedure. Controls in these experiments were fungus grown for 16 hours without macrophages and macrophages without fungal spores. Fungal RNA was isolated using RiboPure Yeast Kit (Thermo Fisher) and macrophage RNA was isolated with the RNeasy Mini kit (Qiagen), according to the manufacturers' instructions. The concentration and purity of RNA samples were assessed using a NanoDrop spectrophotometer and Agilent 2100 bioanalyzer. A total of 681 L. corymbifera genes were differentially expressed during the co-infection with macrophages, with 629 down- (data not shown) and 52 up-regulated (Table 4.1). In order to systematically analyse the function of the differentially expressed genes (DEG), GO enrichment analyses were performed using FungiFun2. Overall design: 6 samples in total: Time point 0 with 3 replicates of Lcor strain JMRC:SF:09682; Time point 16 h with 3 replicates of Lcor strain JMRC:SF:09682 in coculture with murine alveolar macrophage cell line MH-S