ABSTRACT: Transcript abundance comparison between BALB/c ears inoculated with Leishmania mexicana and Leishmania mexicana plus promastigote secretory gel (PSG)
Project description:To determine the modulation of gene expression of Leishmania mexicana(M379)-inoculated BALB/c ears in the presence of promastigote secretory gel (PSG) A genome-wide transcriptional analysis was performed by comparing the gene expression profiles of Leishmania mexicana- inoculated BALB/c ears and Leishmania mexicana plus PSG BALB/c ears. Leishmania mexicana amastigotes were purified from mouse cutaneous lesions and transformed in vitro in metacycic promastigotes (MT). After 6, 24 and 48 hours, ears were collected and processed for RNA extraction. Three Biological replicates per condition were run.
Project description:To determine the modulation of gene expression of Leishmania mexicana(M379)-inoculated BALB/c ears in the presence of promastigote secretory gel (PSG)
Project description:We examined the Leishmania mexicana transcriptome to identify differentially regulated mRNAs using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8156 predicted coding regions revealed 288 genes (3.5% of all genes) whose steady-state mRNA levels meet criteria for differential regulation between promastigotes and lesion-derived amastigotes. Interestingly, sample comparison of promastigotes to axenic amastigotes resulted in only 17 genes (0.2%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of an increase in the magnitude of the transcript levels in cells under axenic conditions. The expression data for a subset of genes was validated by quantitative PCR. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Leishmania may represent an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance. Keywords: RNA expression profiling
Project description:Skin from Balb/c mice after Leishmania amazonensis infection. Mice were infected through their footpads with the promastigote form of the protozoon
Project description:Skin from Balb/c mice after Leishmania major infection. Promastigote forms of the protozoon were inoculated in mice footpads. The skin biopsies were analyzed after 60 days of infection.
Project description:Transcriptional analyses of L. infantum promastigote compared to L. infantum intracellular amastigote, and L. major promastigote compared to L. major intracellular amastigote The full-genome DNA microarray includes one 70mer-oligonucleotide probe for each gene of L. infantum and for each gene of L.major LV39 Keywords: stage-specific comparison Leishmania infantum: Two-condition experiment, promastigote stage vs amastigote stage. Six biological replicates for each stage, independently grown and harvested. One replicate per array Leishmania major: Two-condition experiment, promastigote stage vs amastigote stage. Four biological replicates for each stage, independently grown and harvested. One replicate per array
Project description:The study of extracellular vesicles has become an incredibly important field of study, but the inherent heterogeneity of these vesicles continues to make their study challenging. The genetic variability and well-documented protocols for the growth and vesicle isolation from Leishmania parasites provide a unique opportunity to compare the heterogeneity of different populations secreted by Leishmania clones. Leishmania mexicana was cultured on solid SDM agar plates and 8 clonal colonies were selected. The EVs collected from the liquid cultures of these 8 clones were assessed by NTA, TEM, and proteomic analysis. We found that all 8 clonal L. mexicana cultures were visually indistinguishable from each other and had similar growth rate, and these physical similarities extended to their EVs. However, proteomic analysis reveals that the EVs collected have unique protein profiles compared to each other and EVs isolated from a heterogeneous liquid culture of L. mexicana. We selected 3 clonal EVs for further mouse infection experiments and found that EVs from CL7 L. mexicana consistently caused reduced footpad swelling in C57BL6 mice footpads compared to EVs from CL1, CL8, and heterogenous L. mexicana. This trend was not observed when infecting Balb/C mice with the parasites alone, with only CL1 L. mexicana causing significantly increased infection, nor in C57BL6 mice with all clonal L. mexicana parasites causing a similar infection. Our results together show that EVs isolated from different clonal colonies of L. mexicana have distinct differences in protein cargo which can lead to varying outcomes on Leishmania infection. Further evaluation will be needed to determine the underlying mechanisms behind this and verify that differences observed in infectivity are directly caused by variations between our L. mexicana clones, especially genetic sequencing and immunoblotting to validate our results.
Project description:Leishmania are protists (class: Kinetoplastida) with a single multifunctional flagellum used for motility, attachment to the sand fly vector and sensory functions. To discover the protein composition of the L. mexicana promastigote flagellum, we fractionated L. mexicana promastigotes by mechanical shearing and differential centrifugation into four fractions: detergent soluble (Fs) and insoluble (Fi) fractions of isolated flagella and detergent soluble (Cs) and insoluble fractions (Ci) of deflagellated cell body remnants. A label-free quantitation method (SINQ; Trudgian et al., 2011, Proteomics 10.1002) was used to identify proteins enriched in each of the four fractions. This PRIDE upload contains .RAW, .MGF and .XML files, as well as the SINQ quantification output file “SINQ_raw_data” and the genome sequence annotation used for protein identification “MFiebig_20140619” (Fiebig et al., 2015, PLoS Pathogens II:e1005186). XML files are named SUB7467; MSS10859. .RAW and .MGF files are structured as follows (for each fraction there are eight files, corresponding to individual gel pieces): “Ci: C131121_030; C131121_031; C131121_032; C131121_033; C131121_034; C131121_035; C131121_036; C131121_037”, “Cs: C131121_040; C131121_041; C131121_042; C131121_043; C131121_044; C131121_045; C131121_046; C131121_047”, “Fi: C131121_010; C131121_011; C131121_012; C131121_013; C131121_014; C131121_015; C131121_016; C131121_017”, “Fs: C131121_020; C131121_021; C131121_022; C131121_023; C131121_024; C131121_025; C131121_026; C131121_027”.
