Label-free proteomics and systems biology analysis of mycobacterial phagosomes in dendritic cells and macrophages

Abstract

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.

Figure 1. Clustering analysis of the six phagosome and two whole cell lysate samples based on the abundance profiles of the 1001 quantified proteins

Φ – phagosome. WC – whole cell lysate.

Figure 2. Phagosome and antigen presentation pathways

The rounded rectangular text boxes represent the phagosome components that are a protein or protein complex. All the shown phagosome components except γ-secretase are annotated in the KEGG Phagosome Pathway (www.genome.jp). The bold fonts indicate that a protein is detected in this study for the phagosome component.

Figure 3. Clustering of the six phagosome samples based on the phagosome and antigen presentation pathway (panel a) or the antigen processing and presentation pathway (panel b)

The numbers in parenthesis in panel b indicate the rankings of the proteins in a GSEA leading edge analysis. In panel a, both the samples and proteins were clustered. In panel b, the samples were clustered while the proteins were ordered by their rankings in the GSEA analysis. The duplicate runs for each sample are shown.