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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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Susceptibility to extrinsic proteases of TlyA expressing M. smegmatis (A,B) and E. coli (C,D):M. smegmatis/TlyA (C) and E. coli/TlyA (D) were treated with Proteinase K and the resultant samples were processed for 12% SDS–PAGE and detected with anti-TlyA antibody. P and S indicate pellet and supernatant obtained at indicated times in minutes. The antibodies used for the detection of the blot are indicated on the right side. The lane marked with TlyA indicates purified TlyA as control. The (B,D) respectively represent the hemolytic activity of the Proteinase K treated bacteria, shown in (A,C). The labels are indicated below the bars. Proteinase K treated bacteria.(7 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis by measuring the absorbance at 540 nm of RBC free supernatant.
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Figure 2: Susceptibility to extrinsic proteases of TlyA expressing M. smegmatis (A,B) and E. coli (C,D):M. smegmatis/TlyA (C) and E. coli/TlyA (D) were treated with Proteinase K and the resultant samples were processed for 12% SDS–PAGE and detected with anti-TlyA antibody. P and S indicate pellet and supernatant obtained at indicated times in minutes. The antibodies used for the detection of the blot are indicated on the right side. The lane marked with TlyA indicates purified TlyA as control. The (B,D) respectively represent the hemolytic activity of the Proteinase K treated bacteria, shown in (A,C). The labels are indicated below the bars. Proteinase K treated bacteria.(7 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis by measuring the absorbance at 540 nm of RBC free supernatant.

Mentions: We next examined for exposed amino acid segments of TlyA upon expression in M. smegmatis and E. coli with the help of limited proteolysis by Proteinase K. In this approach, we have incubated the bacteria expressing the TlyA with and without Proteinase K and equal aliquots of bacteria were then analyzed by SDS-PAGE followed by immunoblotting. In principle, even small surface-exposed loops/amino-acid segments are expected to be cleaved upon exposure to Proteinase K. As shown in Figures 2A,C, the intensities of the bands of the full-length TlyA, associated with M. smegmatis (Figure 2A) and E. coli (Figure 2C), were reduced by more than 50% upon Proteinase K treatment. In contrast, the intensity of the intracellular GroEL remained constant over the same period implying that the Proteinase K treatment of the bacteria has not resulted in digestion/degradation of intracellular proteins. Consistent with this observation, the Proteinase K digestion has diminished the contact dependent hemolytic activity of the M. smegmatis (Figure 2B) and E. coli (Figure 2D) associated TlyA, which is a characteristic property of TlyA expressing bacteria (King et al., 1993; Wren et al., 1998; Rahman et al., 2010). It is clear from the figure that the hemolysis has significantly reduced in 10 min. Thus, it is likely that the amino-acid segments of TlyA are exposed at the bacterial surface of M. smegmatis and E. coli and accessible to Proteinase K.


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)

Susceptibility to extrinsic proteases of TlyA expressing M. smegmatis (A,B) and E. coli (C,D):M. smegmatis/TlyA (C) and E. coli/TlyA (D) were treated with Proteinase K and the resultant samples were processed for 12% SDS–PAGE and detected with anti-TlyA antibody. P and S indicate pellet and supernatant obtained at indicated times in minutes. The antibodies used for the detection of the blot are indicated on the right side. The lane marked with TlyA indicates purified TlyA as control. The (B,D) respectively represent the hemolytic activity of the Proteinase K treated bacteria, shown in (A,C). The labels are indicated below the bars. Proteinase K treated bacteria.(7 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis by measuring the absorbance at 540 nm of RBC free supernatant.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Susceptibility to extrinsic proteases of TlyA expressing M. smegmatis (A,B) and E. coli (C,D):M. smegmatis/TlyA (C) and E. coli/TlyA (D) were treated with Proteinase K and the resultant samples were processed for 12% SDS–PAGE and detected with anti-TlyA antibody. P and S indicate pellet and supernatant obtained at indicated times in minutes. The antibodies used for the detection of the blot are indicated on the right side. The lane marked with TlyA indicates purified TlyA as control. The (B,D) respectively represent the hemolytic activity of the Proteinase K treated bacteria, shown in (A,C). The labels are indicated below the bars. Proteinase K treated bacteria.(7 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis by measuring the absorbance at 540 nm of RBC free supernatant.
Mentions: We next examined for exposed amino acid segments of TlyA upon expression in M. smegmatis and E. coli with the help of limited proteolysis by Proteinase K. In this approach, we have incubated the bacteria expressing the TlyA with and without Proteinase K and equal aliquots of bacteria were then analyzed by SDS-PAGE followed by immunoblotting. In principle, even small surface-exposed loops/amino-acid segments are expected to be cleaved upon exposure to Proteinase K. As shown in Figures 2A,C, the intensities of the bands of the full-length TlyA, associated with M. smegmatis (Figure 2A) and E. coli (Figure 2C), were reduced by more than 50% upon Proteinase K treatment. In contrast, the intensity of the intracellular GroEL remained constant over the same period implying that the Proteinase K treatment of the bacteria has not resulted in digestion/degradation of intracellular proteins. Consistent with this observation, the Proteinase K digestion has diminished the contact dependent hemolytic activity of the M. smegmatis (Figure 2B) and E. coli (Figure 2D) associated TlyA, which is a characteristic property of TlyA expressing bacteria (King et al., 1993; Wren et al., 1998; Rahman et al., 2010). It is clear from the figure that the hemolysis has significantly reduced in 10 min. Thus, it is likely that the amino-acid segments of TlyA are exposed at the bacterial surface of M. smegmatis and E. coli and accessible to Proteinase K.

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