Project description:The main objective of this study is to identify the list of genes differentially expressed between infected with Leishmania braziliensis and non-infected macrophage cultures based on gene expression microarray profiling The dataset is comprised by the expression profile of 6 samples from three independent experiments and each experiment had three technical replicates. 3 of the 6 samples were U937 derived macrophages infected by Leishmania braziliensis and the other 3 were U937 derived macrophages without infection with Leishmania braziliensis. A total of 18 microarrays analysis were performed.
Project description:Trained immunity is a phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. This study profiled the transcriptome of human monocytes trained with Leishmania braziliensis and then re-exposed to lipopolysaccharide.
Project description:The main objective of this study is to identify the list of genes differentially expressed between infected with Leishmania braziliensis and non-infected macrophage cultures based on gene expression microarray profiling
Project description:We functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L. braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L. braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Using anti-MMA antibodies, we performed an IP experiment to identify MMA proteins in the parental line, single PRMT1 knockout, PRMT1/PRMT7 double knockout and PRMT1-Addback parasites. The results indicate that R-methylation is modulated across lifecycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions. Linked publication: https://doi.org/10.1101/2021.09.22.461376.
Project description:Throughout their life cycle, Leishmania parasites, causative agents of the life-threatening leishmaniasis, alternate between the phlebotomine vector and the mammalian host, encountering significant environmental changes that require rapid adaptation in gene expression for survival. In the absence of conventional promoters for RNA polymerase II and given the polycistronic nature of transcription, gene expression regulation in Leishmania occurs primarily at the post-transcriptional level. Although putative regulatory non-coding RNAs (ncRNAs) have been detected in the transcriptomes of L. braziliensis and other trypanosomatids, their functional role remains poorly understood. Recognizing the critical role of RNA structure in cellular systems, we sought to identify evolutionarily conserved RNA structures. We performed a genome-wide alignment of L. braziliensis genome with closely related genomes, predicting 142 conserved RNA structures, among which 38 overlap with previously identified ncRNA transcripts. One of the putative ncRNAs with a conserved structure, a 407-nucleotide long ncRNA (lncRNA45), was previously shown to be differentially expressed across the lifecycle stages of L. braziliensis. Here, we conducted a functional characterization of lncRNA45 using a knockout line, demonstrating that the transcript is crucial for parasite fitness. Reintroduction of the original lncRNA45 sequence restored the fitness of the knockout cell line to parental levels, whereas a sequence carrying a single nucleotide substitution in the structured locus did not. In vitro pull-down assays with both the original and modified versions of lncRNA45 revealed differences in the profile of lncRNA-binding proteins. Our results indicate that lncRNA45 is enrolled cellular processes of L. braziliensis promastigotes, and this activity, along with its interaction with proteins, depends on its secondary structure. This study underscores the importance of structured lncRNAs in Leishmania and advocates for further research into their regulatory roles and therapeutic potential.