Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages

Abstract

Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells.

Figure 1

Mtb-infected macrophages release EVs that contain host exosome markers and Mtb molecules. Macrophages were infected with Mtb for 4 h, washed, and incubated for 20 h in vesicle production medium. EVs were purified from the conditioned culture medium by differential ultracentrifugation and analyzed for composition and size. (A) Size distribution of EVs released by uninfected and Mtb-infected (MOI =5) macrophages. Purified EVs were analyzed by tunable resistive pulse sensing using the Izon qNano system. (B) EVs released by uninfected (MOI = 0) and Mtb-infected macrophages (MOI = 3 or 6) were analyzed by Western blotting for exosome markers (CD9, CD63, and GAPDH) or Mtb molecules. The polyclonal anti-Mtb Ab primarily detects the lipoglycans LAM and LM. Results are representative of at least three independent experiments.

Figure 2

Immunoelectron microscopy of EVs released by Mtb-infected macrophages reveals distinct subpopulations of vesicles bearing Mtb or exosome markers. EVs were purified from the culture supernatants of Mtb-infected macrophages, fixed, and analyzed by whole mount electron microscopy. (A) EVs appear as vesicles with a cup-like morphology. (B) Single immunogold labeling of EVs for MHC-II, a component of exosomes released by macrophages. (C), Single immunogold labeling of EVs using the anti-Mtb Ab. (D) Dual immunogold labeling of EVs with antibodies against MHC-II (10 nm gold particles) and Mtb (5 nm gold particles). (E) Triple immunogold labeling of EVs with antibodies against Mtb, MHC-II, and CD9 (5, 10 and 18 nm gold particles, respectively). All scale bars are 100 nm.

Figure 3

Immunofluorescence microscopy reveals that EVs from Mtb-infected macrophages are comprised of subpopulations with largely non-overlapping distributions of host and bacterial markers. Purified EVs from Mtb-infected macrophages were fixed on coverslips, stained for host and Mtb markers, and visualized by immunofluorescence microscopy. Each dot represents an individual vesicle. Colocalization of two markers within a vesicle is indicated by white arrowheads (right panels). (A) Immunofluorescence staining for two host markers (CD9, green; CD63, red). Images reveal significant overlap between the two host markers. (B) Immunofluorescence staining for Mtb (green) and a host marker (CD63 or CD9, red). Images reveal little overlap between host and Mtb markers. Images are representative of more than three independent experiments. (C) Colocalization analysis from two independent experiments (values represent mean ± SD.) The quantitation is expressed as the percentage of each vesicle population (Mtb+, CD63+, or CD9+ EVs; x-axis labels) that is positive for a second marker (indicated by labels at the top of the graph).

Figure 4

EVs subpopulations bearing exosome and Mtb molecules can be separated by sucrose density gradient centrifugation. Macrophages were infected for 4 h with Mtb (MOI = 5), washed and incubated for 20 h. EVs were purified by differential ultracentrifugation from the conditioned medium, overlaid on a continuous 0.2-1.8 M sucrose gradient, and centrifuged for 19 h at 110,000 × g. Gradient fractions were collected and analyzed by Western blotting with Abs specific for exosome or Mtb molecules. Results are representative of three independent experiments.

Figure 5

Release of BMVs from intracellular Mtb is dependent on bacterial viability. (A) Macrophages were incubated for 4 h at an MOI or MOI equivalent of 5 with live Mtb, irradiated Mtb (60,000 rad with a ¹³⁷Cs source; viability reduced by 99.87%), or heat-killed Mtb (80° C for 30 min; viability reduced by 100%). Macrophages were washed and incubated for 20 h. EVs were purified from the conditioned medium and assessed for host or Mtb markers. Results are representative of three independent experiments. (B, C) RAW264.7 macrophages were infected for 2 h at an MOI or MOI equivalent of 20 with live Mtb (B) or heat-killed Mtb (C), washed to remove extracellular bacteria, and then disrupted by Dounce homogenization to release intracellular bacteria. Bacteria were isolated from macrophage homogenates on Percoll gradients and then analyzed by transmission electron microscopy.

Figure 6

TLR2 signaling and cytokine production are induced by BMVs but not exosomes from Mtb-infected macrophages. (A) EVs released from Mtb-infected cells were purified by differential centrifugation and separated on sucrose density gradients. Fractions were pooled (#2-4, no vesicle control; #5-7, exosomes; #8-10 BMVs), concentrated, exchanged into DMEM culture medium, and analyzed for host and bacterial markers by Western blotting. Results are representative of two independent experiments. (B) HEK cells transfected with human TLR2 and CD14 were treated with the indicated sucrose gradient fractions for 24 h. TLR2 activation was assessed by ELISA for the production of IL-8. Results are the average of two replicates and are representative of two independent experiments. (C) Murine macrophages were incubated with the pooled sucrose gradient fractions for 24 h and TNF-α production was assessed by ELISA. Results are the average of two replicates and are representative of three independent experiments.

Figure 7

Proposed models for the trafficking of Mtb molecules into EVs and their release from Mtb-infected macrophages. (A) Existing model: extracellular release of Mtb molecules within exosomes. This model predicts that Mtb cell wall components are released from Mtb within the phagosome, inserted into host membranes, and transported to MVEs, where they are incorporated into inclusion vesicles (alternatively inclusion vesicles could be formed by analogous mechanisms within phagosomes). Exosomes bearing Mtb molecules are then released by exocytosis upon MVE fusion with the plasma membrane. (B) Revised model: extracellular release of Mtb molecules within BMVs. This model predicts that Mtb employs the release of BMVs as a mechanism to export Mtb molecules such as lipoglycans and lipoproteins. BMVs are produced by intracellular Mtb within the phagosome, dispersed through the endocytic system of the infected cell, and exocytosed in a manner similar to exosomes.