Polyomic analyses for rational antileishmanial vaccine development: a role for membrane transporters?

Kabli, Abdulbaset Mohammad Mostafa (2021) Polyomic analyses for rational antileishmanial vaccine development: a role for membrane transporters? PhD thesis, University of Glasgow.

Abstract

Leishmania parasites are transmitted by the bite of a sandfly vector, and have the ability to subvert the host immune system and adopt sophisticated strategies to develop and survive within the mammalian host. Leishmania parasites survive within macrophages, inside a parasitophorous vacuole and cause leishmaniasis. The molecular communication between host and parasite decides the outcome of infection, but is incompletely understood. Additionally, there is no vaccine currently available against human leishmaniasis, existing chemotherapies are toxic, and drug resistance is prevalent. However, leishmaniasis is vaccinable, as evidenced by acquired immunity and the availability of canine vaccines. Live attenuated Leishmania parasites can be exploited as potential vaccines that deliver prolonged immune stimulation from the native parasite niche.
We have compared genotype and gene expression of an attenuated Leishmania mexicana line with a virulent, isogenic wild-type precursor. The attenuated L. mexicana (H-line) was developed by in vitro culturing the parasites under gentamicin pressure. The H-line parasites of various Leishmania species have demonstrated potential as vaccines in rodents models and dogs, and a L. major H-line is currently undergoing phase 2 and phase 3 trials in humans. Therefore, we aim to identify key virulence factors and to explore the potential of the attenuated H-line as a vaccine candidate.
This study was conducted on promastigotes of L. mexicana and involved comprehensive polyomics approaches to elucidate the molecular mechanism that underpins attenuation in Leishmania parasites. Log phase promastigotes of wildtype and the gentamicin-attenuated H-line were grown in parallel and harvested for comparative polyomics analyses. For genomics, DNA was extracted and converted into DNA library for next generation sequencing. For transcriptomics, RNA was isolated and converted into a library of cDNA molecules for cluster generation and RNA sequencing. For proteomics, protein extracts were labelled using 6-plex TMT and analyzed with LC-MS/MS. For metabolomics, finger-print and foot-print metabolites were extracted with chloroform/methanol/water (1:3:1) and analyzed with LC-MS. Integration of polyomics data was carried out based on the gene ontology of the modulated omics products. Selected genes were targeted by CRISPR-Cas9 to assess their individual role in attenuation.
Genomics data of the attenuated H-line revealed the presence of many gene variants and an increase of copy number in chromosomes 16 and 30, which were triploid and tetraploid, respectively. We found 481 transcripts were differentially expressed, 18 proteins differentially expressed, and 26 identified metabolites were differentially represented in the attenuated H-line Leishmania. Statistical significance thresholds in these datasets were assigned for FC ≥1.5 and FDR ≤0.05. Most of the differentially expressed proteins and transcripts were metabolic enzymes, and the majority of the differentially expressed metabolites were substrates involved in nucleotide, carbohydrate and amino acid metabolism. Based on gene ontology, correlation of polyomics datasets revealed that nucleotide metabolism was significantly altered in gentamicin-attenuated Leishmania. Interestingly, several transmembrane nutrient transporters were modulated. Nucleoside (NT1) and nucleobase (NT3) transporters were significantly down regulated in proteomics analysis. Modulation of gene expression, observed through polyomics analyses, may relate to gentamicin selection, either directly through drug selection or as an adaptive consequence of drug selection.
The H-line L. mexicana and LmexΔNT3 cells become more sensitive to allopurinol, a hypoxanthine analogue, at a lower concentration (EC50= 39.1 ± 6.35 and 14.1 ± 0.65 µM, respectively, comparing to the wild-type cells (EC50= 136 ± 4.23 µM). In contrast, LmexΔNT1 cells were less sensitive to tubercidin, an adenosine analogue, (EC50= 7.74 ± 0.21 µM) comparing to the wild-type cells (EC50= 0.76 ± 0.01 µM). This suggests that NT1 and NT3 transporters may contribute to Leishmania virulence. The attenuated H-line invaded but was unable to survive in vitro within THP-1 macrophage-like cells, whereas wild-type parasites were able to survive and multiply. Moreover, findings showed that the H-line, LmexΔNT1, and LmexΔNT3 L. mexicana harboured less than 2 parasites per an infected THP-1 cell compared to the wild-type parasites which harboured about 4 parasites per an infected THP-1 cell. Important concerns about the possibility of reversion to a virulent phenotype must be further addressed before live vaccines can be approved for use in the field.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Please contact [e-mail] or [e-mail] to request a copy while the thesis is on embargo.
• Keywords: Leishmania, mexicana, Leishmaniasis, drug resistance, attenuation, gentamicin, vaccine development, polyomics, genomics, transcriptomics, proteomics, metabolomics, TMT, NGS, LC-MS, sensitivity, infection, CRISPR-Cas9, molecular parasitology, membrane transporters, NT1, NT3, BT1, neglected tropical diseases, NTDs, kinetoplastid.
• Subjects: Q Science > Q Science (General); Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH345 Biochemistry; Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Parasitology
• Supervisor's Name: Burchmore, Dr Richard and Michael, Prof Barrett
• Date of Award: 2021
• Embargo Date: 22 February 2022
• Depositing User: Mr Abdulbaset Kabli
• Unique ID: glathesis:2021-82030
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 Feb 2021 08:37
• Last Modified: 24 Feb 2021 08:37
• Thesis DOI: 10.5525/gla.thesis.82030
• URI: https://theses.gla.ac.uk/id/eprint/82030

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