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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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(A) Expression of TlyA in M. smegmatis:M. smegmatis/TlyA and M. smegmatis (2 × 107) were processed for SDS-PAGE (12%) and stained with Coomassie brilliant blue G-250. The lanes indicated with M, MtbTlyA, Ms/MtbTlyA, and Ms respectively represent Molecular weight markers (kD), Purified MtbTlyA protein from E. coli, whole cell lysate of M. smegmatis expressing MtbTlyA, wild-type M. smegmatis. The MtbTlyA isolated from E. coli expression is 10 amino acids shorter than the M. smegmatis expressed protein. Boxed region (MW range ~21–45 kD) was subjected to LC-MS analysis (Supplementary Tables 1–4). (B) Immunoblot of TlyA expressing M. smegmatis:M. smegmatis (2 × 107) or pMyNT-MtbTlyA were processed for SDS-PAGE and probed with anti-TlyA antibody. The lane indications are as follows: TlyA: Purified MtbTlyA from E. coli. Ms/TlyA: Whole cell lysate of M. smegmatis expressing MtbTlyA; Ms: wild-type M. smegmatis. (C) MsTlyA is undetectable in M. smegmatis:M. smegmatis wild type was fractionated as described in methods section and examined for the presence of MsTlyA (MSMEG_3751) by using the same antibody used in (B). The lane markings represent the samples as follows: TlyA, Purified MtbTlyA from E. coli; CD, cells debris obtained after 3000 g value; CW, cell-wall fraction obtained after 27000 g; MEM, pellet of the membrane fraction obtained after 100,000 g centrifugation; SOL, supernatant of the 100,000 g centrifugation. The top panel was developed with anti-TlyA antibody, middle panel represents the identification of MsHBHA obtained with anti-HBHA antibody (BEI resources), and the bottom panel represents the MsGroEL obtained with anti-GroEL antibody (BEI resources). (D) Detection of tlyA transcript: RT-PCR of mRNA from MtbTlyA transformed and non-transformed M. smegmatis was done to detect the tlyA transcript as described in methods section. The lanes indicate: M: DNA ladder; Lanes 1–5 respectively represent the RT-PCR products using the DNA templates of pMyNT/tlyA vector (positive control), cDNA of M. smegmatis/tlyA, cDNA of non-transformed M. smegmatis, RNA of transformed M. smegmatis/tlyA and RNA of non-transformed M. smegmatis. (E) Visualization of TlyA on M. marinum: Thin smears of M. marinum were visualized in confocal microscope after staining with rabbit anti-TlyA antibody and Rhodamine-anti-rabbit-IgG. The top and bottom panels represent the staining of M. marinum with normal rabbit serum (NRS) and immune rabbit serum specific to MtbTlyA (IRS) respectively. The panels, left, middle, and right represent the signal positive to MmTlyA, DAPI for bacterial staining and merged panels of MmTlyA and DAPI. Panels shown are a representative of one the several independent visualizations. (F,G) Visualization of TlyA in M. smegmatis (F) and E. coli (G): The bacteria and the constructs are indicated on the left side of the panels. The primary antibody used is rabbit anti-TlyA antibody and secondary antibody was Rhodamine-anti-rabbit-IgG. The middle panels represent the DAPI staining. The merged panels are overlap of Rhodamine and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. (H) Dual stating of TlyA transformed M. smegmatis: TlyA was visualized with anti-TlyA antibody and Rhodamine anti-rabbit antibody (TlyA-Rh). HBHA was visualized with mouse monoclonal anti-HBHA antibody and with FITC-anti-mouse-IgG (HBHA-FITC). DAPI labeled panels was obtained by staining of the bacteria with DAPI. The merged panels are overlap of TlyA-Rh, HBHA-FITC, and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. Intracellular staining of TlyA transformed M. smegmatis: TlyA transformed M. smegmatis, without (middle) and with (bottom) permeabilization, were stained for GroEL with mouse monoclonal anti-GroEL antibody and anti-mouse-IgG-FITC. DAPI labeled panel indicates the staining of the bacteria DAPI. The merged panels are overlap of FITC and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the three independent visualizations.
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Figure 1: (A) Expression of TlyA in M. smegmatis:M. smegmatis/TlyA and M. smegmatis (2 × 107) were processed for SDS-PAGE (12%) and stained with Coomassie brilliant blue G-250. The lanes indicated with M, MtbTlyA, Ms/MtbTlyA, and Ms respectively represent Molecular weight markers (kD), Purified MtbTlyA protein from E. coli, whole cell lysate of M. smegmatis expressing MtbTlyA, wild-type M. smegmatis. The MtbTlyA isolated from E. coli expression is 10 amino acids shorter than the M. smegmatis expressed protein. Boxed region (MW range ~21–45 kD) was subjected to LC-MS analysis (Supplementary Tables 1–4). (B) Immunoblot of TlyA expressing M. smegmatis:M. smegmatis (2 × 107) or pMyNT-MtbTlyA were processed for SDS-PAGE and probed with anti-TlyA antibody. The lane indications are as follows: TlyA: Purified MtbTlyA from E. coli. Ms/TlyA: Whole cell lysate of M. smegmatis expressing MtbTlyA; Ms: wild-type M. smegmatis. (C) MsTlyA is undetectable in M. smegmatis:M. smegmatis wild type was fractionated as described in methods section and examined for the presence of MsTlyA (MSMEG_3751) by using the same antibody used in (B). The lane markings represent the samples as follows: TlyA, Purified MtbTlyA from E. coli; CD, cells debris obtained after 3000 g value; CW, cell-wall fraction obtained after 27000 g; MEM, pellet of the membrane fraction obtained after 100,000 g centrifugation; SOL, supernatant of the 100,000 g centrifugation. The top panel was developed with anti-TlyA antibody, middle panel represents the identification of MsHBHA obtained with anti-HBHA antibody (BEI resources), and the bottom panel represents the MsGroEL obtained with anti-GroEL antibody (BEI resources). (D) Detection of tlyA transcript: RT-PCR of mRNA from MtbTlyA transformed and non-transformed M. smegmatis was done to detect the tlyA transcript as described in methods section. The lanes indicate: M: DNA ladder; Lanes 1–5 respectively represent the RT-PCR products using the DNA templates of pMyNT/tlyA vector (positive control), cDNA of M. smegmatis/tlyA, cDNA of non-transformed M. smegmatis, RNA of transformed M. smegmatis/tlyA and RNA of non-transformed M. smegmatis. (E) Visualization of TlyA on M. marinum: Thin smears of M. marinum were visualized in confocal microscope after staining with rabbit anti-TlyA antibody and Rhodamine-anti-rabbit-IgG. The top and bottom panels represent the staining of M. marinum with normal rabbit serum (NRS) and immune rabbit serum specific to MtbTlyA (IRS) respectively. The panels, left, middle, and right represent the signal positive to MmTlyA, DAPI for bacterial staining and merged panels of MmTlyA and DAPI. Panels shown are a representative of one the several independent visualizations. (F,G) Visualization of TlyA in M. smegmatis (F) and E. coli (G): The bacteria and the constructs are indicated on the left side of the panels. The primary antibody used is rabbit anti-TlyA antibody and secondary antibody was Rhodamine-anti-rabbit-IgG. The middle panels represent the DAPI staining. The merged panels are overlap of Rhodamine and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. (H) Dual stating of TlyA transformed M. smegmatis: TlyA was visualized with anti-TlyA antibody and Rhodamine anti-rabbit antibody (TlyA-Rh). HBHA was visualized with mouse monoclonal anti-HBHA antibody and with FITC-anti-mouse-IgG (HBHA-FITC). DAPI labeled panels was obtained by staining of the bacteria with DAPI. The merged panels are overlap of TlyA-Rh, HBHA-FITC, and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. Intracellular staining of TlyA transformed M. smegmatis: TlyA transformed M. smegmatis, without (middle) and with (bottom) permeabilization, were stained for GroEL with mouse monoclonal anti-GroEL antibody and anti-mouse-IgG-FITC. DAPI labeled panel indicates the staining of the bacteria DAPI. The merged panels are overlap of FITC and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the three independent visualizations.

Mentions: LC-MS analysis of the boxed region in Figure 1A was performed as per the mass spectrometry facility of NCCS using protocols published earlier (Målen et al., 2007; Prados-Rosales et al., 2011; Reddy et al., 2015).


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)

(A) Expression of TlyA in M. smegmatis:M. smegmatis/TlyA and M. smegmatis (2 × 107) were processed for SDS-PAGE (12%) and stained with Coomassie brilliant blue G-250. The lanes indicated with M, MtbTlyA, Ms/MtbTlyA, and Ms respectively represent Molecular weight markers (kD), Purified MtbTlyA protein from E. coli, whole cell lysate of M. smegmatis expressing MtbTlyA, wild-type M. smegmatis. The MtbTlyA isolated from E. coli expression is 10 amino acids shorter than the M. smegmatis expressed protein. Boxed region (MW range ~21–45 kD) was subjected to LC-MS analysis (Supplementary Tables 1–4). (B) Immunoblot of TlyA expressing M. smegmatis:M. smegmatis (2 × 107) or pMyNT-MtbTlyA were processed for SDS-PAGE and probed with anti-TlyA antibody. The lane indications are as follows: TlyA: Purified MtbTlyA from E. coli. Ms/TlyA: Whole cell lysate of M. smegmatis expressing MtbTlyA; Ms: wild-type M. smegmatis. (C) MsTlyA is undetectable in M. smegmatis:M. smegmatis wild type was fractionated as described in methods section and examined for the presence of MsTlyA (MSMEG_3751) by using the same antibody used in (B). The lane markings represent the samples as follows: TlyA, Purified MtbTlyA from E. coli; CD, cells debris obtained after 3000 g value; CW, cell-wall fraction obtained after 27000 g; MEM, pellet of the membrane fraction obtained after 100,000 g centrifugation; SOL, supernatant of the 100,000 g centrifugation. The top panel was developed with anti-TlyA antibody, middle panel represents the identification of MsHBHA obtained with anti-HBHA antibody (BEI resources), and the bottom panel represents the MsGroEL obtained with anti-GroEL antibody (BEI resources). (D) Detection of tlyA transcript: RT-PCR of mRNA from MtbTlyA transformed and non-transformed M. smegmatis was done to detect the tlyA transcript as described in methods section. The lanes indicate: M: DNA ladder; Lanes 1–5 respectively represent the RT-PCR products using the DNA templates of pMyNT/tlyA vector (positive control), cDNA of M. smegmatis/tlyA, cDNA of non-transformed M. smegmatis, RNA of transformed M. smegmatis/tlyA and RNA of non-transformed M. smegmatis. (E) Visualization of TlyA on M. marinum: Thin smears of M. marinum were visualized in confocal microscope after staining with rabbit anti-TlyA antibody and Rhodamine-anti-rabbit-IgG. The top and bottom panels represent the staining of M. marinum with normal rabbit serum (NRS) and immune rabbit serum specific to MtbTlyA (IRS) respectively. The panels, left, middle, and right represent the signal positive to MmTlyA, DAPI for bacterial staining and merged panels of MmTlyA and DAPI. Panels shown are a representative of one the several independent visualizations. (F,G) Visualization of TlyA in M. smegmatis (F) and E. coli (G): The bacteria and the constructs are indicated on the left side of the panels. The primary antibody used is rabbit anti-TlyA antibody and secondary antibody was Rhodamine-anti-rabbit-IgG. The middle panels represent the DAPI staining. The merged panels are overlap of Rhodamine and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. (H) Dual stating of TlyA transformed M. smegmatis: TlyA was visualized with anti-TlyA antibody and Rhodamine anti-rabbit antibody (TlyA-Rh). HBHA was visualized with mouse monoclonal anti-HBHA antibody and with FITC-anti-mouse-IgG (HBHA-FITC). DAPI labeled panels was obtained by staining of the bacteria with DAPI. The merged panels are overlap of TlyA-Rh, HBHA-FITC, and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. Intracellular staining of TlyA transformed M. smegmatis: TlyA transformed M. smegmatis, without (middle) and with (bottom) permeabilization, were stained for GroEL with mouse monoclonal anti-GroEL antibody and anti-mouse-IgG-FITC. DAPI labeled panel indicates the staining of the bacteria DAPI. The merged panels are overlap of FITC and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the three independent visualizations.
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Figure 1: (A) Expression of TlyA in M. smegmatis:M. smegmatis/TlyA and M. smegmatis (2 × 107) were processed for SDS-PAGE (12%) and stained with Coomassie brilliant blue G-250. The lanes indicated with M, MtbTlyA, Ms/MtbTlyA, and Ms respectively represent Molecular weight markers (kD), Purified MtbTlyA protein from E. coli, whole cell lysate of M. smegmatis expressing MtbTlyA, wild-type M. smegmatis. The MtbTlyA isolated from E. coli expression is 10 amino acids shorter than the M. smegmatis expressed protein. Boxed region (MW range ~21–45 kD) was subjected to LC-MS analysis (Supplementary Tables 1–4). (B) Immunoblot of TlyA expressing M. smegmatis:M. smegmatis (2 × 107) or pMyNT-MtbTlyA were processed for SDS-PAGE and probed with anti-TlyA antibody. The lane indications are as follows: TlyA: Purified MtbTlyA from E. coli. Ms/TlyA: Whole cell lysate of M. smegmatis expressing MtbTlyA; Ms: wild-type M. smegmatis. (C) MsTlyA is undetectable in M. smegmatis:M. smegmatis wild type was fractionated as described in methods section and examined for the presence of MsTlyA (MSMEG_3751) by using the same antibody used in (B). The lane markings represent the samples as follows: TlyA, Purified MtbTlyA from E. coli; CD, cells debris obtained after 3000 g value; CW, cell-wall fraction obtained after 27000 g; MEM, pellet of the membrane fraction obtained after 100,000 g centrifugation; SOL, supernatant of the 100,000 g centrifugation. The top panel was developed with anti-TlyA antibody, middle panel represents the identification of MsHBHA obtained with anti-HBHA antibody (BEI resources), and the bottom panel represents the MsGroEL obtained with anti-GroEL antibody (BEI resources). (D) Detection of tlyA transcript: RT-PCR of mRNA from MtbTlyA transformed and non-transformed M. smegmatis was done to detect the tlyA transcript as described in methods section. The lanes indicate: M: DNA ladder; Lanes 1–5 respectively represent the RT-PCR products using the DNA templates of pMyNT/tlyA vector (positive control), cDNA of M. smegmatis/tlyA, cDNA of non-transformed M. smegmatis, RNA of transformed M. smegmatis/tlyA and RNA of non-transformed M. smegmatis. (E) Visualization of TlyA on M. marinum: Thin smears of M. marinum were visualized in confocal microscope after staining with rabbit anti-TlyA antibody and Rhodamine-anti-rabbit-IgG. The top and bottom panels represent the staining of M. marinum with normal rabbit serum (NRS) and immune rabbit serum specific to MtbTlyA (IRS) respectively. The panels, left, middle, and right represent the signal positive to MmTlyA, DAPI for bacterial staining and merged panels of MmTlyA and DAPI. Panels shown are a representative of one the several independent visualizations. (F,G) Visualization of TlyA in M. smegmatis (F) and E. coli (G): The bacteria and the constructs are indicated on the left side of the panels. The primary antibody used is rabbit anti-TlyA antibody and secondary antibody was Rhodamine-anti-rabbit-IgG. The middle panels represent the DAPI staining. The merged panels are overlap of Rhodamine and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. (H) Dual stating of TlyA transformed M. smegmatis: TlyA was visualized with anti-TlyA antibody and Rhodamine anti-rabbit antibody (TlyA-Rh). HBHA was visualized with mouse monoclonal anti-HBHA antibody and with FITC-anti-mouse-IgG (HBHA-FITC). DAPI labeled panels was obtained by staining of the bacteria with DAPI. The merged panels are overlap of TlyA-Rh, HBHA-FITC, and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the several independent visualizations. Intracellular staining of TlyA transformed M. smegmatis: TlyA transformed M. smegmatis, without (middle) and with (bottom) permeabilization, were stained for GroEL with mouse monoclonal anti-GroEL antibody and anti-mouse-IgG-FITC. DAPI labeled panel indicates the staining of the bacteria DAPI. The merged panels are overlap of FITC and DAPI channels The white bar represents 2 μm. Panels shown are a representative of one the three independent visualizations.
Mentions: LC-MS analysis of the boxed region in Figure 1A was performed as per the mass spectrometry facility of NCCS using protocols published earlier (Målen et al., 2007; Prados-Rosales et al., 2011; Reddy et al., 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.

Show MeSH
Related in: MedlinePlus