The role of metabolic enzymes in virulence and immunopathogenesis of Leishmania infection
| dc.contributor.author | Onwah, Somtochukwu Stella | |
| dc.contributor.examiningcommittee | Thomas Murooka (Immunology) | |
| dc.contributor.examiningcommittee | Soussi-Gounni, Abdelilah (Immunology) | |
| dc.contributor.examiningcommittee | Duan, Kangmin (Oral Biology) | |
| dc.contributor.examiningcommittee | Ramanathan, Sheela (University de Sherbrooke) | |
| dc.contributor.supervisor | Uzonna, Jude | |
| dc.date.accessioned | 2025-01-10T14:23:36Z | |
| dc.date.available | 2025-01-10T14:23:36Z | |
| dc.date.issued | 2024-12-22 | |
| dc.date.submitted | 2024-12-22T16:21:37Z | en_US |
| dc.date.submitted | 2025-01-09T20:23:41Z | en_US |
| dc.degree.discipline | Immunology | |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | |
| dc.description.abstract | Leishmaniasis is a neglected tropical disease affecting approximately a million people yearly. The disease is caused by intracellular protozoan parasites that belong to the genus Leishmania. Different Leishmania species cause different clinical forms of the disease, which can present as self-healing cutaneous lesions or often fatal visceral disease that affects the liver, spleen and bone marrow. Unfortunately, there is yet no approved vaccine against human leishmaniasis. Although some drugs are effective, treatment often results in serious drug-mediated toxicity and side effects, and there is increasing evidence of drug resistance. However, vaccination is possible because people who recover from the disease are immune to reinfection. Knowledge of the antigens that induce protective immunity in infected hosts is critical for designing effective vaccines and therapeutic strategies against the disease. Dihydrolipoyl dehydrogenase (DLD) and Phosphoenolpyruvate carboxykinase (PEPCK) are key metabolic enzymes in numerous eukaryotic cells, including Leishmania and play critical roles in metabolism. This present study aims to assess the impact of DLD and PEPCK deficiency on parasite virulence and host immune response. Using the CRISPR-Cas 9 gene editing tool DLD and PEPCK gene in L. major and L. donovani, respectively, were deleted. The impact of deficiency of these enzymes on parasite survival, infectivity and host immune response were assessed. The gene-deficient parasites demonstrated reduced proliferation in axenic cultures and inside infected macrophages in vitro. Mice infected with DLD-deficient parasites had no observable lesions and harboured significantly lower parasite burden compared to their WT or complementary add-back control-infected animals. Interestingly, DLD deficient L. major infected mice mounted blunted immune responses compared to their WT and complementary addback infected control counterpart mice. Vaccination with DLD deficient parasites induced strong protective responses upon virulent L. major rechallenge, and this was associated with strong IFN- response. The ability for PEPCK-deficient L. donovani to differentiate into their infective forms was not compromised in vitro. Although their infectivity in macrophages were compromised in vitro, these parasites maintained their virulence and pathology in vivo. In mice infected with PEPCK deficient L. donovani, immune responses in the liver and spleens were relatively unaltered apart from increased TNF production in the liver. Interestingly, the infectivity of PEPCK KO parasites in Kupffer cells in vitro was compromised compared to their WT or AB parasite controls, suggesting that the in vivo microenvironments may predispose PEPCK-deficient parasites to a compensatory mechanism that enables their proliferation. Collectively, these findings show that DLD is a critical metabolic enzyme for intracellular survival of L. major and targeting this molecule could be a viable option for controlling the disease. Although PEPCK-deficient L. donovani is attenuated in vitro but not in vivo, they may not be a suitable live attenuated vaccine candidate against visceral leishmaniasis. | |
| dc.description.note | February 2025 | |
| dc.identifier.uri | http://hdl.handle.net/1993/38773 | |
| dc.language.iso | eng | |
| dc.rights | open access | en_US |
| dc.subject | CRISPR/Cas9 | |
| dc.subject | metabolic enzymes | |
| dc.subject | Leishmaniasis | |
| dc.title | The role of metabolic enzymes in virulence and immunopathogenesis of Leishmania infection | |
| dc.type | doctoral thesis | en_US |
| local.subject.manitoba | no |