Differential gene expression in DEVH1 knock-in Leishmania infantum promastigotes

The DEAD/H RNA helicase LINF_220021200 (DEVH1) gene from Leishmania infantum (Kinetoplastida:Trypanosomatidae) was cloned in the pTEX expression plasmid vector for trypanosomatids. Leishmania infantunm promastigotes were transfected and a knock-in L. infantum promastigote cell line was selected with geneticin (G418). A pTEX control promastigote line was also generated. Then, three independent biological replicate cultures of each pTEX-DEVH1 and pTEX promastigote lines were performed in the presence of the selective agent. The parasites were harvested on day 7 (stationary phase). Total mRNA samples were obtained. Cyanine dye-labelled samples were obtained from the knock-in and the control line (Cy5 and Cy3, respectively) and they were hybridized with custom whole-genome L. infantum DNA microarrays. This platform is included in GEO (GPL6781) and has also been repeatedly used in different experiments from 2009. Hybridization analysis allowed for finding differentially expressed genes due to the effect of induced over-expression of the DEVH1-encoding gene in the knock-in promastigote line compared to the control line. Genes involved in parasite infectivity and survival such as the HASP/SHERP gene cluster and an amastin gene or redox homeostasis genes are significantly down-regulated in the pTEX-DEVH1 knock-in promastigote line, whereas genes related to growth are up-regulated. This is in agreement with previous experimental data supporting that L. infantum DEVH1 knock-in promastigotes are able to recover the growth rate when stress conditions are removed. Six samples were analyzed. Three were biological replicates of stationary phase promastigotes of the pTEX-DEVH1 L. infantum line. All other three samples were biological replicates of stationary phase promastigotes of the pTEX L. infantum control line. The samples were cDNA labelled with Cy5 and Cy3, respectively. Normalization of fluorescence intensity values was performed by the LOWESS per pin algorithm. Student`s t-test was conducted for statistical inference, including FDR adjustment. Please note that the 'pTEX-DEVH1 vs. pTEX' sample represents three replicates (3 raw data files; Analyzed_dataset_pTEX_DEVH1_vs_pTEX_Li.txt) and averaged data across arrays (i.e. mean of normalized fold change data).