Transcript abundance comparison between BALB/c ears inoculated with Leishmania mexicana and Leishmania mexicana plus promastigote secretory gel (PSG)
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ABSTRACT: 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:Transcript abundance comparison between BALB/c ears inoculated with Leishmania mexicana and Leishmania mexicana plus 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. This experiment appears as Fig. 1 of the associated publication. Keywords: RNA expression profiling
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:Chronic Otitis Media (OM) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute OM pathogen, is known to activate inflammation and mucin expression in vitro and in animal models of OM. The goals of this study were to: examine expression profiling epithelial effects of NTHi challenge in murine middle ears. We used microarrays to detail examine the global programme of gene expression underlying epithelial effects of NTHi challenge in murine middle ears during this study. Weekly transtympanic inoculation of Balb/c mice with 300 µg/ml of NTHi lysates vs saline was performed. Bacteria were grown on chocolate agar at 37ºC in 5% CO2 overnight and inoculated in brain heart infusion (BHI) broth supplemented with 3.5 mg of nicotinamide adenine dinucleotide per ml. After overnight incubation, bacteria were subcultured into 5 ml of fresh brain heart infusion (BHI) and upon reaching log phase growth, NTHi were washed and suspended in phosphate-buffered saline (PBS) followed by sonication for lysis. Three transtympanic inoculation of 6 Balb/c mice middle ears (3 animals, 6 ears) with 50 uL of 300 ug/ml of NTHi bacterial lysate and 6 Balb/c mice middle ears (3 animals, 6 ears) with 50 uL of 1X phosphate buffered saline (PBS) were carried out weekly over 4 weeks (injection on days 7, 14, and 21). On day 28, the mice were euthanized and their bullae harvested. Expression microarray analysis was performed at 1 and 7 days. Microarray findings were validated in independent animal samples and in a cultured murine middle ear epithelial cell (mMEEC) line.
Project description:We used Illumina sequencing of poly-A selected RNA of Leishmania mexicana (WHO strain MNYC/BZ/62/M379) culture-adapted promastigotes (PRO), axenic amastigotes (AXA) and intracellular amastigotes (AMA) in mouse bone marrow derived macrophages (BMDM), 24h after infection, to map 5' and 3' ends of Leishmania transcripts and determine transcript abundances. The AMA samples were prepared from total RNA of infected macrophages thus containing a mixture of leishmanial and murine RNA transcripts. We also sequenced poly-A selected RNA from uninfected BMDMs. Three biological replicates per sample.
Project description:Affinity purification of deubiquitinase DUB2 from Leishmania mexicana promastigotes. Myc tagged DUB2 was purified from cross linked Leishmania parasites in 3 independent replicates and compared to affinity purification of an unrelated myc tagged protein by label free quantification.