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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) Immunofluorescence visualization of TlyA in ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains were immuno-stained with anti-TlyA antibody and visualized with Rhodamine conjugated anti-rabbit antibody IgG. Left side panels show the staining for TlyA (red). Middle panels show the DAPI staining of bacteria (blue) and the right panels show the merged with red and blue channels. The bar represents 2 μm. The panels are a representative of one of the three independent visualizations. (B) Contact dependent hemolysis of TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA transformed and non-transformed M. smegmatis (2 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis, in comparison to the water lysed RBC (bar marked with 1) by measuring the absorbance at 540 nm of RBC free supernatant.
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Figure 4: (A) Immunofluorescence visualization of TlyA in ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains were immuno-stained with anti-TlyA antibody and visualized with Rhodamine conjugated anti-rabbit antibody IgG. Left side panels show the staining for TlyA (red). Middle panels show the DAPI staining of bacteria (blue) and the right panels show the merged with red and blue channels. The bar represents 2 μm. The panels are a representative of one of the three independent visualizations. (B) Contact dependent hemolysis of TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA transformed and non-transformed M. smegmatis (2 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis, in comparison to the water lysed RBC (bar marked with 1) by measuring the absorbance at 540 nm of RBC free supernatant.

Mentions: The immunoblot in Figure 3C shows an unambiguous presence of MtbTlyA in the membrane fraction of M. smegmatis upon its expression. Interestingly, the MtbTlyA is also seen in the membrane fractions of M. smegmatis deficient in ΔTatA (Figure 3D), ΔTatC (Figure 3E) and ΔSecA2 (Figure 3F). It is relevant to note that SecA1 deficiency is lethal for M. smegmatis and hence, could not be studied (Braunstein et al., 2001). We have also examined for HBHA and GroEL as internal controls of this fractionation. While the HBHA is always restricted to cell-wall fraction, the GroEL is found only in soluble fraction than membrane fraction, which validates our fractionation attempt. After having visualized the TlyA in M. smegmatis, we next performed sub-cellular fractionation of E. coli/TlyA to separate outer-membrane, periplasmic, inner-membrane, and cytosolic fractions by well established protocols (Wai et al., 2003b). The TlyA is present in inner-membrane, periplasmic space and outer membrane (Figure 3G). Under the same conditions, the GroEL, β-lactamase and GFP were observed in cytosolic, periplasmic and cytosolic fractions which again support our fractionation attempt. The small periplasmic presence of GFP could be due to osmotic shock in the procedure. Hence, the presence of TlyA in the membrane fractions of M. smegmatis or E. coli is not due to artifact of expression but a specific sequestration might be responsible since membrane fractions of both H37Rv, M. marinum also exhibit an unambiguous presence of TlyA. In support of all the above observations, we could easily see the presence of TlyA on the surface of ΔTatA, ΔTatC, and ΔSecA2 deficient strains of M. smegmatis while the mock vector-transformed bacteria did not exhibit any positive fluorescence staining as seen in Figure 4A In support of this observation, all the TlyA expressing ΔTatA, ΔTatC, and ΔSecA2 knockout strains have exhibited contact dependent hemolytic activity (Figure 4B). The data shown in Figure 4B clearly let us infer that there is no significant difference in hemolytic activity among the TlyA expressing strains. Hence, the TlyA does not seem to depend on either Tat or Sec pathways for translocation to cell-wall of M. smegmatis.


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) Immunofluorescence visualization of TlyA in ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains were immuno-stained with anti-TlyA antibody and visualized with Rhodamine conjugated anti-rabbit antibody IgG. Left side panels show the staining for TlyA (red). Middle panels show the DAPI staining of bacteria (blue) and the right panels show the merged with red and blue channels. The bar represents 2 μm. The panels are a representative of one of the three independent visualizations. (B) Contact dependent hemolysis of TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA transformed and non-transformed M. smegmatis (2 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis, in comparison to the water lysed RBC (bar marked with 1) by measuring the absorbance at 540 nm of RBC free supernatant.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: (A) Immunofluorescence visualization of TlyA in ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains were immuno-stained with anti-TlyA antibody and visualized with Rhodamine conjugated anti-rabbit antibody IgG. Left side panels show the staining for TlyA (red). Middle panels show the DAPI staining of bacteria (blue) and the right panels show the merged with red and blue channels. The bar represents 2 μm. The panels are a representative of one of the three independent visualizations. (B) Contact dependent hemolysis of TlyA expressing ΔTatA, ΔTatC and ΔSecA2 deficient strains of M. smegmatis: TlyA transformed and non-transformed M. smegmatis (2 × 107) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis, in comparison to the water lysed RBC (bar marked with 1) by measuring the absorbance at 540 nm of RBC free supernatant.
Mentions: The immunoblot in Figure 3C shows an unambiguous presence of MtbTlyA in the membrane fraction of M. smegmatis upon its expression. Interestingly, the MtbTlyA is also seen in the membrane fractions of M. smegmatis deficient in ΔTatA (Figure 3D), ΔTatC (Figure 3E) and ΔSecA2 (Figure 3F). It is relevant to note that SecA1 deficiency is lethal for M. smegmatis and hence, could not be studied (Braunstein et al., 2001). We have also examined for HBHA and GroEL as internal controls of this fractionation. While the HBHA is always restricted to cell-wall fraction, the GroEL is found only in soluble fraction than membrane fraction, which validates our fractionation attempt. After having visualized the TlyA in M. smegmatis, we next performed sub-cellular fractionation of E. coli/TlyA to separate outer-membrane, periplasmic, inner-membrane, and cytosolic fractions by well established protocols (Wai et al., 2003b). The TlyA is present in inner-membrane, periplasmic space and outer membrane (Figure 3G). Under the same conditions, the GroEL, β-lactamase and GFP were observed in cytosolic, periplasmic and cytosolic fractions which again support our fractionation attempt. The small periplasmic presence of GFP could be due to osmotic shock in the procedure. Hence, the presence of TlyA in the membrane fractions of M. smegmatis or E. coli is not due to artifact of expression but a specific sequestration might be responsible since membrane fractions of both H37Rv, M. marinum also exhibit an unambiguous presence of TlyA. In support of all the above observations, we could easily see the presence of TlyA on the surface of ΔTatA, ΔTatC, and ΔSecA2 deficient strains of M. smegmatis while the mock vector-transformed bacteria did not exhibit any positive fluorescence staining as seen in Figure 4A In support of this observation, all the TlyA expressing ΔTatA, ΔTatC, and ΔSecA2 knockout strains have exhibited contact dependent hemolytic activity (Figure 4B). The data shown in Figure 4B clearly let us infer that there is no significant difference in hemolytic activity among the TlyA expressing strains. Hence, the TlyA does not seem to depend on either Tat or Sec pathways for translocation to cell-wall of M. smegmatis.

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