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Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence.

Kumar S, Mittal E, Deore S, Kumar A, Rahman A, Krishnasastry MV - Front Cell Infect Microbiol (2015)

Bottom Line: However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments.Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence.Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Membrane Biology, National Centre for Cell Science, Savitribai Phule Pune University Pune, India.

ABSTRACT
The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

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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.
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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.

Mentions: Both gram negative and gram positive bacteria secrete Outer Membrane Vesicles (OMV) and Membrane Vesicles (MV) respectively (Wai et al., 2003a; Prados-Rosales et al., 2011). We, therefore, examined whether we can detect TlyA in MV secreted by M. smegmatis and OMV secreted by E. coli. The supernatants used for the identification of TlyA in the membrane vesicles was thoroughly assessed for the absence of viable bacteria by plating an aliquot on appropriate plate after filtration through 0.45 μm membrane. As shown in Figure 5A (MV from M. smegmatis) and Figure 5C (OMV from E. coli), the presence of TlyA is unambiguous in both MV and OMV fractions. In this regard, we have used DnaK in case of MV from M. smegmatis and β-lactamase in case of OMV from E. coli to authenticate our vesicle preparations. Both MV and OMV shown above contain active TlyA since they are able to lyse rabbit RBC as seen in Figures 5B,D. We have also attempted to examine these vesicles by negative staining in transmission electron microscopy. We have found small outer membrane vesicles surrounding the intact bacterium with gold particles (Figure 5E right panel) while the non-transformed bacteria did not exhibit any gold particles on its periphery (Figure 5E left panel). We have also examined the purified MV and OMV by immune-gold labeling and negative staining. The EM micrographs of MV of M. smegmatis/TlyA (Figure 5F top right panel) and OMV of E. coli/TlyA (Figure 5F bottom right panel) have shown gold particles at the periphery of the vesicles while the non-transformed MV (Figure 5F top left panel) or OMV (Figure 5F bottom left panel) did not exhibit any gold particles in their periphery. These observations suggest that the bacterially expressed TlyA is capable of reaching the extra-cellular milieu using a vesicle mediated transport.


Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence.

Kumar S, Mittal E, Deore S, Kumar A, Rahman A, Krishnasastry MV - Front Cell Infect Microbiol (2015)

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.
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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.
Mentions: Both gram negative and gram positive bacteria secrete Outer Membrane Vesicles (OMV) and Membrane Vesicles (MV) respectively (Wai et al., 2003a; Prados-Rosales et al., 2011). We, therefore, examined whether we can detect TlyA in MV secreted by M. smegmatis and OMV secreted by E. coli. The supernatants used for the identification of TlyA in the membrane vesicles was thoroughly assessed for the absence of viable bacteria by plating an aliquot on appropriate plate after filtration through 0.45 μm membrane. As shown in Figure 5A (MV from M. smegmatis) and Figure 5C (OMV from E. coli), the presence of TlyA is unambiguous in both MV and OMV fractions. In this regard, we have used DnaK in case of MV from M. smegmatis and β-lactamase in case of OMV from E. coli to authenticate our vesicle preparations. Both MV and OMV shown above contain active TlyA since they are able to lyse rabbit RBC as seen in Figures 5B,D. We have also attempted to examine these vesicles by negative staining in transmission electron microscopy. We have found small outer membrane vesicles surrounding the intact bacterium with gold particles (Figure 5E right panel) while the non-transformed bacteria did not exhibit any gold particles on its periphery (Figure 5E left panel). We have also examined the purified MV and OMV by immune-gold labeling and negative staining. The EM micrographs of MV of M. smegmatis/TlyA (Figure 5F top right panel) and OMV of E. coli/TlyA (Figure 5F bottom right panel) have shown gold particles at the periphery of the vesicles while the non-transformed MV (Figure 5F top left panel) or OMV (Figure 5F bottom left panel) did not exhibit any gold particles in their periphery. These observations suggest that the bacterially expressed TlyA is capable of reaching the extra-cellular milieu using a vesicle mediated transport.

Bottom Line: However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments.Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence.Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Membrane Biology, National Centre for Cell Science, Savitribai Phule Pune University Pune, India.

ABSTRACT
The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

Show MeSH
Related in: MedlinePlus