Proteomic analysis of detergent resistant membrane domains during early interaction of macrophages with rough and smooth Brucella melitensis

Abstract

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide the basis for novel ways of treating or diagnosing Brucellosis.

Figure 1. Growth of Brucella in Mono Mac 6 cells and attachment of Brucella melitensis to Mono Mac 6 cells.

Figure 1 A shows the survival of B. melitensis 16 M in Mono Mac 6 cells over 96 hours at 37°C. Figure 1 B depicts the internalization of rough and smooth B. melitensis and B. abortus strains into Mono Mac 6 cells at 37°C for up to 20 minutes. B. abortus strains appear to enter Mono Mac 6 cells slightly faster (within about 1 minute) than B. melitensis strains (within about 4 minutes) as indicated by the lowest CFU numbers which represent the time when all bacteria have become intracellular (extracellular bacteria have been killed by gentamicin and intracellular bacteria are not multiplying yet). The confocal images in Figures 1 C and D show the adherence of RFP-expressing B. melitensis to Mono Mac 6 cells at 1 minute (C) and 2 minutes (D) upon exposure at 4°C. Mono Mac 6 cells not exposed to Brucella are shown in Figure 1 E.

Figure 2. Processing of DRMDs for the comparative proteomic analysis using the iTRAQ method.

The schematic shows the work flow for processing of DRMD samples from uninfected and infected cells for the quantitative mass spectrometry analysis using the iTRAQ method. Interfering lipids and sucrose were removed using Vivaspin filters before DRMD samples were reduced with dithiothreitol, followed by alkylation with iodoacetamide and trypsinization. These treatments are routinely used in the preparation of samples for labeling with iTRAQ reagents for the mass spectrometric analysis were found to effectively kill Brucella.

Figure 3. Networks of proteins based on enriched gene ontology functions.

Response networks were constructed with Network Express by using the corresponding iTRAQ proteins as seed nodes from the four scenarios described in the legend for Tables 1 and 2 together with the quantitative values from the iTRAQ experiments. Networks were drawn with Cytoscape .A: VTRM1 IN + no change; B: VTRM1 OUT + no change; C: 16M IN + no change, D: 16M OUT + no change. IN  =  +, ++, +++ or ++++. OUT  =  -, —, --- or ----. Nodes: red: up-regulated (IN) blue: down-regulated (OUT) white: no change (0) green: no data. The more red/blue, the higher the fold-change. Full red/blue for the highest bracket of the iTRAQ scores. Edges: black: high score (“strong/likely connection”) yellow: low score (“weak/less likely connection”).