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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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Subcellular fractionation of M. tuberculosis (A) M. marinum (B), TlyA expressing M. smegmatis WT (C), ΔTatA (D), ΔTatC (E), ΔSecA2 (F), and E. coli/TlyA (G): The bacterial strains are indicated at the top of each panel. For all panels, the antibodies used for detection are indicated on the right side. Purified TlyA from E. coli was used as a marker for identification of TlyA band in all blots. The samples used for (A) were obtained from BEI resources. WC and MEM represent whole cell lysate and membrane fraction respectively. In (B–F), the lane markings CD, CW, MEM, and SOL respectively represent cell debris obtained after 3000 g, cell-wall after 27,000 g and membrane pellet fraction obtained after 100,000 g and soluble fraction of 100,000 g (supernatant). In panel G, the C, P, OM, and IM respectively represent the cytosolic, periplasmic, outer-membrane, and inner-membrane fractions. The panels are a representative of one of the three independent experiments.
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Figure 3: Subcellular fractionation of M. tuberculosis (A) M. marinum (B), TlyA expressing M. smegmatis WT (C), ΔTatA (D), ΔTatC (E), ΔSecA2 (F), and E. coli/TlyA (G): The bacterial strains are indicated at the top of each panel. For all panels, the antibodies used for detection are indicated on the right side. Purified TlyA from E. coli was used as a marker for identification of TlyA band in all blots. The samples used for (A) were obtained from BEI resources. WC and MEM represent whole cell lysate and membrane fraction respectively. In (B–F), the lane markings CD, CW, MEM, and SOL respectively represent cell debris obtained after 3000 g, cell-wall after 27,000 g and membrane pellet fraction obtained after 100,000 g and soluble fraction of 100,000 g (supernatant). In panel G, the C, P, OM, and IM respectively represent the cytosolic, periplasmic, outer-membrane, and inner-membrane fractions. The panels are a representative of one of the three independent experiments.

Mentions: The data in Figures 3A,B respectively show the presence of MtbTlyA and MmTlyA in the membrane fractions of H37Rv and M. marinum respectively. It is relevant to mention here that the whole cell lysate and membrane fractions, shown in Figure 3A, were obtained from BEI resources and serve as an independent verification. As expected, the MmTlyA is also detected in the membrane fraction as seen Figure 3B.


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)

Subcellular fractionation of M. tuberculosis (A) M. marinum (B), TlyA expressing M. smegmatis WT (C), ΔTatA (D), ΔTatC (E), ΔSecA2 (F), and E. coli/TlyA (G): The bacterial strains are indicated at the top of each panel. For all panels, the antibodies used for detection are indicated on the right side. Purified TlyA from E. coli was used as a marker for identification of TlyA band in all blots. The samples used for (A) were obtained from BEI resources. WC and MEM represent whole cell lysate and membrane fraction respectively. In (B–F), the lane markings CD, CW, MEM, and SOL respectively represent cell debris obtained after 3000 g, cell-wall after 27,000 g and membrane pellet fraction obtained after 100,000 g and soluble fraction of 100,000 g (supernatant). In panel G, the C, P, OM, and IM respectively represent the cytosolic, periplasmic, outer-membrane, and inner-membrane fractions. The panels are a representative of one of the three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543871&req=5

Figure 3: Subcellular fractionation of M. tuberculosis (A) M. marinum (B), TlyA expressing M. smegmatis WT (C), ΔTatA (D), ΔTatC (E), ΔSecA2 (F), and E. coli/TlyA (G): The bacterial strains are indicated at the top of each panel. For all panels, the antibodies used for detection are indicated on the right side. Purified TlyA from E. coli was used as a marker for identification of TlyA band in all blots. The samples used for (A) were obtained from BEI resources. WC and MEM represent whole cell lysate and membrane fraction respectively. In (B–F), the lane markings CD, CW, MEM, and SOL respectively represent cell debris obtained after 3000 g, cell-wall after 27,000 g and membrane pellet fraction obtained after 100,000 g and soluble fraction of 100,000 g (supernatant). In panel G, the C, P, OM, and IM respectively represent the cytosolic, periplasmic, outer-membrane, and inner-membrane fractions. The panels are a representative of one of the three independent experiments.
Mentions: The data in Figures 3A,B respectively show the presence of MtbTlyA and MmTlyA in the membrane fractions of H37Rv and M. marinum respectively. It is relevant to mention here that the whole cell lysate and membrane fractions, shown in Figure 3A, were obtained from BEI resources and serve as an independent verification. As expected, the MmTlyA is also detected in the membrane fraction as seen Figure 3B.

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