Quantitative proteomic profiling of host-pathogen interactions: the macrophage response to Mycobacterium tuberculosis lipids

Abstract

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen possessing a complex mixture of cell wall lipids that are thought to modulate the activities of host macrophages. In this study, we employed two state-of-the-art quantitative proteomic approaches, metabolic labeling SILAC and chemical isobaric tagging iTRAQ, to study changes in macrophage protein expression in response to exposure to M. tuberculosis lipids. From a total of 1286 proteins identified, 463 were discovered by both isotope-labeling strategies at a high consistency, and the rest of proteins were detected by only one of the two approaches. Upon exposure to mycobacterial cell wall lipids, 166 macrophage proteins showed differential expression. These included proteins involved in the immune response, oxidation and reduction, and vesicle transport, as well as other cellular processes. The response of the macrophage proteome to M. tuberculosis lipids reflects the cell's innate defense mechanisms as well as lipid-induced processes that may benefit the pathogen.

Figure 1

Effects of M. tb lipid extract on TNF-α production of macrophages grown in normal (black bar) and isotope-enriched (white bar) media (containing ¹³C₆-lysine and ¹³C₆, ¹⁵N₄-arginine). Cells were incubated for 24h with different amount of M. tb lipid extract or LPS (100 ng/mL) and the cell-free supernatants were collected for measurements of TNF-α secretion via ELISA. The experiment was repeated in triplicate and error bars indicate the standard deviation of the mean.

Figure 2

Venn diagram of protein identification via either iTRAQ or SILAC approach. Two injection replicates were made for each method, which are indicated by 1 and 2.

Figure 3

Distribution of protein ratios in a log scale. R_iTRAQ and R_SILAC refer to the expression ratio measured by iTRAQ and SILAC for a specific protein. The SD (0.08) reflects a narrow variability.

Figure 4

Relative distribution of macrophage protein subsets regulated by M. tb lipid exposure. Proteins are grouped by their cellular functions. The up- and down-regulated hits are combined in each group. The ‘Miscellaneous’ group consists mostly of proteins of unknown function.

Scheme 1

Schematic of the iTRAQ and SILAC strategy for labeling and quantifying the macrophage proteome under stimulation of a low (S1) or high (S2) dose of M. tb lipid extract.