Systematic lipidomics study of Talaromyces marneffei infection

Abstract

Lipidomics study provides the information of the structure and function of the cellular lipids as well as their interactions with other lipids, metabolites and proteins. This dissertation focuses on the systematic lipidomics studies of Talaromyces (Penicillium) Marneffei (T. marneffei) infection with the combination of liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) techniques. The lipidomics study started with the investigation of the lipid-protein interactions between pro-inflammatory lipid mediators and Mp1p ligand-binding domain 2 (Mp1p-LBD2), which is one of the most critical virulence factors of T. marneffei. Previous studies revealed that Mp1p-LBD2 protein is (1) an important virulence factor of T. marneffei and (2) capable of trapping a number of fatty acids including palmitic acid and arachidonic acid. Mp1p-LBD2 protein forms a specially designed five-helix bundle with a long hydrophobic central cavity to trap fatty acids tightly. We applied the in-vitro pull-down method using recombinant Mp1p-LBD2 protein to prove that arachidonic acid is the dominant cellular target of Mp1p-LBD2 protein. Furthermore, in the cell model, we showed that Mp1p is capable of reducing cellular levels of arachidonic acid and results in the downregulation of arachidonic acid downstream metabolites as well as pro-inflammatory cytokines IL6, and TNF-α. Our study gives insights into the virulence mechanism of Mp1p and its role in the pathogenesis of T. marneffei. Additionally, a number of Mp1p-LBD2 homologues with similar structures and high binding affinity towards fatty acids are found in other pathogenic fungi. This well-characterized virulence mechanism of scavenging pro-inflammatory lipid mediators to dampen the inflammatory response may contribute to a better understanding of fungal pathogenesis. Another part of our research was aimed to carry out the cell-based lipidomics study to highlight significant lipid biomarkers of T. marneffei infection as well as to provide a preliminary understanding of the lipid-cytokine story. We developed a high throughput LC-MS platform for both untargeted and targeted lipidomics study including: (1) efficient lipid extraction protocols; (2) well-developed and advanced LC-MS methods; (3) a lipid library contained over 220 lipid standards with optimized LC-MS conditions. The LC-MS-based lipidomics study of T. marneffei infection identified that linoleic acid, one of the omega-6 fatty acids, was in great demand when the macrophage was infected with T. marneffei. Linoleic acid together with its metabolites 9-HODE and 13-HODE were considered as significant lipid biomarkers involved in T. marneffei infection. Additionally, the lipidomics study on the Mp1 knockout strain T. marneffei infected macrophage again proved that the virulence factor Mp1p protein of T. marneffei did turn down the arachidonic acid metabolism pathway and lead to dampening of inflammatory response of the host. Lipid-cytokine integration studies showed that the increased levels of HODEs were correlated with the increased expression of interleukin-1β. Therefore interleukin-1β may also be considered as an inflammatory marker in T. marneffei infection.