Figure 5.

Presence of TlyA in Membrane Vesicles (MV) of M. smegmatis (A,B) and Outer Membrane Vesicles (OMV) of E. coli (C,D): The MV secreted by M. smegmatis or OMV secreted by E. coli expressing the TlyA were obtained as described in methods section. The presence of TlyA in the vesicles was ascertained by developing respective blots with anti-TlyA antibody (A,C). Hemolytic activity of MV secreted by M. smegmatis (B) and OMV secreted by E. coli (D) was obtained by mixing 20 μg/ml (total protein) of vesicle preparation with 1.5% rabbit RBC. After 24 h of incubation, the absorbance was measured at 540 nm for release for hemoglobin. EM visualization of OMVs: (E) Electron micrograph of a plain and TlyA transformed M. smegmatis (top right panel) and non-transformed M. smegmatis is shown in top left panels that wee stained with 10 nm gold labeled antibody. The vesicle attached to the intact bacterium is shown marked with arrow. The (F) represents the electron micrograph of a purified MV and OMVs. Both MV and OMV show significant deposition of gold particles as shown in the right panels (marked with arrows) while the left panels show no gold particles which represent the MV and OMV of non-transformed bacteria. In all panels the magnification bar represents 200 nm.