Discussion

The intricate interaction between human host and Leishmania is thought to be insightful in understanding the relationship as well as towards chemotherapeutic development. Employing high-resolution mass spectrometry coupled with pulse-chase stable isotope labeling by amino acid in cell culture (pcSILAC) technique, we delved into the investigation of proteome changes in L. mexicana-infected THP-1. Cells were pre-labelled with L-Arg-¹³C₆ and L-Lys-¹³C₆ until isotope incorporation of ≥98% was achieved. Media was then replaced with light Arg and Lys where they are pulsed into cells for 24 and 48 hour. In other words, protein synthesis is ‘chased’ with unlabelled amino acids. At each pulse, the cells were infected with L. mexicana. 2-D-LCMS/MS data revealed a total of 1998 proteins detected in both control and 24 hour infected cells while 2113 proteins detected in 48 hour infected cells. 34% of the quantified proteins were shown to be considerably upregulated in infected cells. These included Ras-related protein, cytoskeleton proteins such as tubulin and myosin, proteins known to be associated with infection such as annexin, carbonic anhydrase and galectin-9. Conversely, proteins that were significantly downregulated after infection included purine nucleoside phosphorylase, superoxide dismutase (Mn), gelsolin, vimentin, Pyruvate kinase PKM, moesin, Calreticulin, ribosomal proteins and actin. Furthermore, we have identified 405 newly-synthesized proteins after 48 hour infection including Toll-like receptor 2 and Interleukin-1. Some of the proteins we have found to be modulated upon Leishmania infection have previously been implicated in this process, while others have not previously been shown to be involved in macrophage:leishmania interactions.