Gedamu, Lashitewdos Santos Meira, Camila2023-02-222023-02-222023-02-17dos Santos Meira, C. (2023). Leishmania donovani cathepsin B: impact on proteins and ncRNAs of small extracellular vesicles, and on infection metabolism (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/115875https://dx.doi.org/10.11575/PRISM/40763Leishmaniasis is a spectrum of chronic diseases caused by protozoan parasites of the Leishmania genus. These parasites can circumvent immune defenses and survive in phagolysosomes of host cells by disrupting signaling processes and cellular functions. Leishmania virulence factors including papain-like cysteine proteases, have been extensively implicated in immune evasion and parasite differentiation. The disruption of the cathepsin B-like cysteine protease of L. donovani was found to attenuate infectivity and induce proteome remodeling in these parasites, affecting the expression of metabolic enzymes and proteins released in extracellular vesicles (EVs). Here, the effects of L. donovani cathepsin B on EV-derived proteins and small non-coding RNAs (ncRNAs), and on metabolism of infected macrophages were investigated. L. donovani cathepsin B wild type (WT), cathepsin B knockout (KO), and episomally complemented knockout (CM) parasites were used to generate comparative proteomic and transcriptomic profiles of small EVs (sEVs), and of metabolites in the spent media of infected U937 cells. LC-MS/MS semi-quantitative analyses revealed the cathepsin B-induced modulation of diverse elements in sEVs, including metabolic enzymes, calpain cysteine proteases, kinases, and translation-related components. Protein-protein networks were remarkable in sEVs and cathepsin B-induced alterations in their proteomes seemed to differ from those previously observed in the parasites, indicating selective packaging of protein cargo in these vesicles. The exploration of the Illumina sequenced small RNAs (18-30nt) from sEVs showed striking abundances of tRNA, rRNA and snoRNA fragments originating from a small subset of genes and with suspected regulatory functions. Moreover, ncRNA genes were differentially expressed in WT and CM sEVs, while KO sEVs exhibited significant expression of coding genes. The bioinformatics predictions of miRNA-like and other regulatory elements for non-annotated transcripts identified in sEVs suggested potential non-canonical regulatory mechanisms in Leishmania. The mass-spectrometry-based metabolomics study indicated extensive differences in the metabolism of macrophages infected with L. donovani WT, KO and CM, especially in nucleotide, energy and carbon metabolisms. Several disturbances appeared to correlate with cathepsin B-induced modulation of metabolic enzymes. Overall, the data suggest the participation of cathepsin B in protein and RNA sorting/packaging into Leishmania EVs and demonstrate the potential mechanisms by which this protease affects host-parasite interaction.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.ProteomicsTranscriptomicsMetabolomicsLeishmaniaMicrobiologyParasitologydoctoral thesis