The role of antibiotic-derived mycobacterial vesicles in tuberculosis pathogenesis

Abstract

Pulmonary tuberculosis (TB) causes progressive and irreversible damage to lung tissue, a damage that may not fully resolve after treatment. Mycobacterial vesicles (MVs), which are poorly understood, may contribute to TB pathology. This study investigated the effects of stress, such as treatment with conventional TB antibiotics rifampicin, isoniazid, ethambutol, or treatment with an antimycobacterial peptide (NZX), on mycobacterial vesiculation. Stress from minimal inhibitory concentrations of antibiotics, or peptide all increased MV formation. Electron microscopy and lipid profiling revealed that these vesicles, about 40 nm in size, were released from the bacterial inner membrane and consisted of apolar lipids. Using mass spectrometry, the study identified key differences in MVs protein cargo dependent on the antibiotic used, especially with ethambutol-induced MVs that contained proteins from several mycobacterial pathways. Additionally, toxicology analysis using different concentrations of MVs on primary human macrophages and the monocytic cells indicated that MVs from the different treatments were not toxic to human cells, however induced specific inflammatory profiles. In conclusion, this study identified mycobacterial vesicles to be a potential contributor to tuberculosis pathology.

Fig. 1

Antibiotic stress induces MVs. (A) EM pictures showing inner membrane vesicle formation (arrows) and aggregation close to the bacteria. (B) Increased magnification showing vesicles approximately 40 nm in diameter and spherical. Some variation in size was observed. Representative images from three analysed batches of vesicles.

Fig. 2

MVs contain nonpolar lipids. (A) TLC separation of lipids showing the non-polar lipids of mycobacterial MVs migrated up the plate. The TLC controls, consisting of polar phospholipids such as phosphatidylinositol (PI), cardiolipin (CL), and digalactosyldiacylglycerol (DGDG) remained stationary at the sample application site on the TLC plate. (B) MS analysis identified apolar trehalose monomycolate (TMM) in mycobacterial MVs from RIF and NZX induced MVs. TMM was not definitively identified in MVs induced by INH or EMB, and no other lipids could be distinctly identified in the MV lipid samples.

Fig. 3

MV protein concentration and variation. (A) Protein concentrations between MV samples were checked prior to analysis and were found to be not significant (p < 0.8395). (B) There was a variation in number of proteins with most proteins induced by EMB (p < 0.0393).

Fig. 4

Differences in antibiotic induced MVs protein cargos. Volcano plots identifying significant difference between antibiotic induced MVs proteins. (A) Difference between RIF and EMB, (B) EMB and INH and (C) RIF and INH. Differences between (D) NZX and INH, (B) NZX and RIF and (C) EMB and NZX. Data are presented are from three separate MV batches.

Fig. 4

Differences in antibiotic induced MVs protein cargos. Volcano plots identifying significant difference between antibiotic induced MVs proteins. (A) Difference between RIF and EMB, (B) EMB and INH and (C) RIF and INH. Differences between (D) NZX and INH, (B) NZX and RIF and (C) EMB and NZX. Data are presented are from three separate MV batches.

Fig. 5

Characteristics of antibiotic induced mycobacterial MVs. Concentration (4 µg/mL (blue) or 40 (orange) µg/mL) related cytotoxicity analysis in primary human macrophages by (A) MTT and in (B) THP-1 cells by Prestoblue (p < 0.05 compared to other treatments). (C) Inflammatory response was measure by NF-κB-activation in THP-1 cells after addition of antibiotic induced MVs (4 µg/mL (blue) or 40 (red) µg/mL) or HKLM as control (13.4 (± 3.4 SD) (n = 3).

Fig. 6

Murine evaluation of TB related lung fibrosis. Immunostaining showing IL-1β expression in lung tissue following TB infection and treatment. Results of the blinded lung inflammatory score. Data are presented as the mean ± SEM (n = 4–5).

Fig. 7

Cytokine profiles from MV stimulated macrophages. Differences in cytokine induction were observed related to MVs from treatment with RIF, INH, EMB and NZX. Both pro-inflammatory and anti-inflammatory responses were induced (n = 3).