Quantitative proteomic responses of macrophages to Leishmania mexicana infection

The dynamics of protein turnover is central to the regulation of protein expression. The steady-state level of a protein is the net outcome of the change in its rate of synthesis and degradation. Different biological states or perturbations cause changes in the expression of specific proteins, which can be assessed by proteomic analysis to reveal links between genotype and phenotype. Unlike other conventional proteomic methods, which measure the amount of proteins in the system at a specific point in time, pulse-chase stable isotope labelling by amino acids in cell culture (pcSILAC) can reveal changes in the rates of protein synthesis and degradation over time. The causative agent of Leishmaniasis, Leishmania, has a digenetic lifestyle involving an extracellular flagellated promastigote living in the mid-gut of the sand fly vector and an aflagellated intracellular amastigote residing in the macrophage of the mammalian host. As they live in a different niche their protein expression could give insight into their adaptation and survival. The intricate interaction between the human host and the Leishmania parasite is key to pathology and may present new targets for chemotherapeutic development. Employing high-resolution mass spectrometry coupled with pulse-chase SILAC technique, we delved into the investigation of proteome changes in L. mexicana-infected macrophages. The first part of the thesis discusses the quantitative proteomic analysis of L. mexicana promastigote and amastigote stage. In this work, stable isotope dimethyl labelling was employed to differentially labelled promastigotes and axenic amastigotes. Our results revealed transformation from promastigote to amastigote were accompanied by: i) reduced glycolytic and gluconeogenesis pathway, ii) increased fatty acid oxidation, iii) increased mitochondrial respiration, iv) reduced expression of proteins that may have flagellar role (e.g. flagellar connector protein, flagellum targeting protein KHARON1), v) reduced stress response proteins, vi) increased protein synthesis, and vii) increased proteolytic proteins. The findings reported here substantially advance our knowledge on the differences of protein expression in different life cycle stage of L. mexicana and could be useful in finding drug targets. Another part of the thesis discusses the establishment and application of pulse-chase SILAC. In this work, a human macrophage-like cell line (THP-1) was grown in media containing L-Arg-13C6 and L-Lys-13C6 until isotope incorporation of >98% was achieved. Media was then replaced with light arginine and lysine so that light amino acids were pulsed into cells for 24 and 48 hours. In other words, protein synthesis is ‘chased’ with unlabelled amino acids. Synchronous with the switch from pulse to chase, the macrophages were infected with L. mexicana. This approach provides the ability to monitor the rates of heavy-label loss, hence determining protein degradation rates and half-lives. At 24-hour post-infection, when compared to mock-infected cells, 2016 proteins were identified, 761 were quantified, and 51 were significantly modulated at p-value < 0.05. Interestingly, proteins involved in glycolysis were markedly downregulated in synthesis after infection while oxidative phosphorylation and fatty acid β-oxidation had increased synthesis, suggesting a subversion of host cell metabolism by Leishmania which proposed to play a key role in microbial growth and persistence. Additionally, pro-apoptotic proteins such as apoptosis regulator BAX and caspase 3 had increased translation in cells infected for 24 hour. This was accompanied by the overexpression of STAT1 which could result in modulation of apoptotic pathways. These characteristics advocate that THP-1 cells most likely exhibit an M2 macrophage phenotype following 24-hour infection. Temporal proteomic data revealed some striking changes in metabolisms of the host at 24 and 48-hour post-infection. After 48 hours of infection, 2104 proteins were identified, and 84 were significantly modulated post-infection at p-value < 0.05. After 48 hours of infection, relative to levels at 24 hours of infection, host cells increased the synthesis of glycolytic enzymes and reduced oxidative phosphorylation synthesis. Further, a total of 400 newly synthesized proteins were selected based on stringent criteria to measure synthesis rates, degradation rate constant (kdeg) and half-lives. These include several ribosomal proteins, pyruvate kinase, L-lactate dehydrogenase, moesin, several glycolytic enzymes such as glucose-6-phosphate isomerase and alpha enolase, gelsolin, galectin-9, catalase and lamin-B. We found that globally kdeg values in THP-1 were low ranging from 0.01 to 0.04 h-1. Our degradation data indicated that proteins involved in mitochondrial related functions (TCA, oxidative phosphorylation) as well as other energy production pathways were more stable and have longer half-lives. For the 400 proteins, the mean half-life for uninfected 24 h, 24 hpi, uninfected 48h and 48 hpi were 21.74 h, 20.51 h, 47.39 h and 47.33 h, respectively. Intriguingly, newly synthesized proteins involved in immune responses, including HLA complexes, were rapidly degraded in infected cells, despite having decreased synthesis rates after 48 hours of infection. Collectively, most proteins in the present study had decreased kdeg and longer half-lives following longer exposure of THP-1 to L. mexicana. Our data show the potential of pulse-chase SILAC to dissect the response of macrophages to Leishmania infection. To our knowledge, no studies have reported the proteome turnover of macrophage in response to Leishmania infection