Characterization of membrane vesicles released by Mycobacterium avium in response to environment mimicking the macrophage phagosome

Abstract

Aim: To investigate the formation of Mycobacterium avium membrane vesicles (MVs) within macrophage phagosomes.

Materials & methods: A phagosome model was utilized to characterize proteomics and lipidomics of MVs. A click chemistry-based enrichment assay was employed to examine the presence of MV proteins in the cytosol of host cells.

Results: Exposure to metals at concentrations present in phagosomes triggers formation of bacterial MVs. Proteomics identified several virulence factors, including enzymes involved in the cell wall synthesis, lipid and fatty acid metabolism. Some of MV proteins were also identified in the cytosol of infected macrophages. MVs harbor dsDNA.

Conclusion: M. avium produces MVs within phagosomes. MVs carry products with potential roles in modulation of host immune defenses and intracellular survival.

Keywords: AHA; bioorthogonal metabolic labeling; macrophages; membrane vesicles; minimal medium; phagosome model.

Figure 1.. Characterization of Mycobacterium avium subsp. hominissuis membrane vesicles.

(A) Scanned electronic microscopy micrographs of MAH104 exposed to minimal media for 2 weeks (a & b) and metal-mix mimicking 24 h phagosome environment (c & d). Scanned electronic microscopy micrographs demonstrate bulges on the bacterial membranes that are about to bud off from the membrane. In addition, bacteria of phagosome model are much elongated when compared with Mycobacterium avium subsp. hominissuis exposed to environmental stress, a phenotype typically seen in intracellular mycobacteria isolated from host cells. Scale bar: 1 μm. (B) Transmission electron microscopy micrographs of purified MV samples with closed membrane structure. MVs are indicated by arrows. (a) Purified membrane vesicle (MV) sample from MAH104 exposed to the minimal media for 2 weeks. (b) Purified MV sample from MAH104 exposed to the metal-mix for 24 h. Scale bar: 300 nm. (C) MAH104 viability assay tested in both minimal media and metal mix for up to 2 weeks. CFUs of bacteria exposed to both minimal media and metal mix show slight decrease over the 2-week course when compared with the 7H9 Middlebrook broth grown control supplemented with 10% oleic acid, albumin, dextrose and catalase. Data were plotted as mean of two independent trials and standard deviation. (D) The size and concentration of membrane vesicles isolated from the minimal media (a) and metal mix (b) were determined by nanoparticle tracking analysis on a Nanosight NS300.

Figure 2.. Membrane vesicle proteomics and conformational studies for secretion of MX proteins.

(A) Venn diagram showing the number of overlapping and unique sets of membrane vesicle cargo proteins found in MAH104 membrane vesicles (MV) released in response to minimal media and metal mix at chosen time points. (B) Functional classification of MV cargo proteins into categories was done by finding the protein homologs of M. tuberculosis H37Rv strain and using the functional categorization available on TubercuList webserver of Institute Pasteur or based on predicted or known function for those Mycobacterium avium proteins that do not match to any proteins of H37Rv strain. Bars show percentage representation of each category in MV cargo proteins of minimal media (a) and metal mix (b). (C) Venn diagram showing common and unique proteins found in MAH104 MVs released in response to metal mix and azidohomoalanine-labeled M. avium subsp. hominissuis presynthesized secreted effectors found in the host cell cytosol at 24 h postinfection of THP-1 macrophages. (D) Analysis of FLAG-tagged MX protein secretion in the cytoplasm of infected macrophages. MAH104 MAV_5152, MAV_2909, MAV_2345, MAV_4365, MAV_2833, MAV_3813, MAV_0740, MAV_2054, MAV_3310, MAV_1082 and MAV_2964 protein overexpression clones were constructed using the pMV261:FLAG vector. (a) Mycobacterium avium subsp. hominissuis clones were mechanically disturbed using bead beater and cleared samples were processed for western-blot analysis to confirm the protein expression within mycobacterial cells. (b) THP-1 cells were infected with MAH104 clones at a multiplicity of infection 1: 10. Precleared and concentrated bacterial and cell lysate-free supernatants were subjected to immunoprecipitation and then western blotting using FLAG-tag antibody. The photon emission means for each protein band are recorded to quantify the signal intensity on the Odyssey Imager (Li-Cor).

Figure 3.. Membrane vesicle lipidomics.

(A) Total/positive-ion chromatograms of membrane vesicles of minimal media and metal mix. (B) Extracted ion chromatograms for individual lipid classes of membrane vesicles of minimal media (a, b, c, d & e) and metal mix (f, g, h, i & j). DAGs: Diacyl gycerols; FFEE: Free fatty acid ethyl esters; FFME: Free fatty acid methyl esters; PEs: Phosphatidylethanolamines; TAGs: Triacylglycerols.

Figure 3.. Membrane vesicle lipidomics.

(A) Total/positive-ion chromatograms of membrane vesicles of minimal media and metal mix. (B) Extracted ion chromatograms for individual lipid classes of membrane vesicles of minimal media (a, b, c, d & e) and metal mix (f, g, h, i & j). DAGs: Diacyl gycerols; FFEE: Free fatty acid ethyl esters; FFME: Free fatty acid methyl esters; PEs: Phosphatidylethanolamines; TAGs: Triacylglycerols.

Figure 4.. Quantification of membrane vesicle-associated DNAs of minimal media and metal mix.

(A) Total, external and internal Membrane vesicle (MV)-associated DNAs were quantified using Quant-iT PicoGreen dsDNA assay (n = 3). Internal DNA was measured by treating intact MV sample with DNase before lysing MVs, whereas a total DNA was measured without treating samples with DNase. *p < 0.05 of internal and external DNA concentrations between the minimal media and metal mix. (B) MV samples were stained with a lipophilic nucleic acid stain SYTO-61, which emits red fluorescence upon DNA binding. Laser confocal images of the DNase treated MVs visualizing the internally associated DNA of minimal media (a) and metal-mix (b). Intact MVs are also seen in the transmitted light images (right panels). Scale bar: 10 μm.

Figure 5.. Freeze-fracture transmission electron microscopy.

Freeze-fracture transmission electron microscopy of Mycobacterium avium subsp. hominissuis-infected THP-1 macrophages reveals numerous membrane vesicles on the cell wall of intracellular bacteria shown by arrows. Several M. avium subsp. hominissuis-containing phagosomes are seen in the cytoplasm of THP-1 cells. N: Nucleus of macrophage; PH: Phagosomes.

Figure 6.. Translocation of membrane vesicles into the cytosol of macrophages.

(A) Approximately 10⁸ Texas Red-labeled MV particles were either directly added to THP-1 macrophages or initially host cells were pretreated with cytochalasin B, monodansylcadaverine, dynasore, methyl-β-cyclodextrin or just DMSO and then added to macrophages. (B) Time-dependent dynamics of intact MV presence within the cytosol of THP-1 cells. Fluorescent readings were recorded with Texas red corresponding filter sets on the Tecan Infinity 200 cytofluorometer. Treatments were made in duplicates and the experiment was repeated three-times. **p < 0.01 between monodansylcadaverine and untreated, DMSO control and all other inhibitor groups. Fluorescent micrographs of THP-1 macrophages exposed with Texas red-labeled MVs in (C) untreated and (D) monodansylcadaverine-pretreated cells after 4-h incubation. Actin filaments are visualized using phalloidin (in green) and nuclei (in blue) with DAPI. MV: Membrane vesicle.