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Research Article

volume 54 issue 12 (december 2020) : 1525-1531,   Doi: 10.18805/ijar.B-3865

Characterization of Putative Type IV Secretion System of Bacillus anthracis

G
Gopal Jee Gopal
A
Awanish Kumar
1C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli-394 350, Surat, Gujarat, India.
Cite article:- Gopal Jee Gopal, Kumar Awanish (2020). Characterization of Putative Type IV Secretion System of Bacillus anthracis. Indian Journal of Animal Research. 54(12): 1525-1531. doi: 10.18805/ijar.B-3865.

ABSTRACT

Background: Bacillus anthracis causes anthrax disease in livestock and occasionally in humans mainly through three toxic proteins: protective antigen, lethal factor and edema factor. Synthesis of the toxic proteins in bacteria and its pathogenic role in the human host is well known. But, how these toxins are secreted from bacterium to the host or in the culture medium is not known. 
Methods: A group of researchers have shown (through in silico prediction) that several genes of B. anthracis have similarity with the genes which codes the subunit of the Type IV Secretion System (T4SS) known in other bacteria. In this study, we have tried to explore the existence of T4SS in B. anthracis using molecular biology tools. 
Result: As an initial lead, three genes (GBAA-pXO1-0064, GBAA-pXO1-0085VirB11 and Bas3783) of B. anthracis T4SS (out of more than 10) were cloned and expressed in E. coli. Among them, gene Bas3783 (VirD4 homolog) is genome encoded therefore; it is sequenced and further characterized for its DNA binding activity and oligomerization. Results obtained from this study indicated the existence of T4SS in B. anthracis.

REFERENCES

  1. Batra, K., Nanda, T., Kumar, A., Kumar, V., Gopal G.J. and Maan, S. (2019). Molecular cloning and expression kinetics of serum interferon stimulated gene for early pregnancy detection. Indian J. Animal Res. (53): 1129-1134.
  2. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M. and van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495-503.
  3. Fronzes, R., Schafer, E., Wang, L., Saibil, H.R., Orlova, E.V. and Waksman, G. (2009). Structure of a type IV secretion system core complex. Science. 323: 266- 268.
  4. Garufi, G., Butler, E., Missiakas, D. (2008). ESAT-6-like protein secretion in Bacillus anthracis. J. Bacteriol. 190(21): 7004-7011.
  5. Gopal, G.J. and Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. Protein. J. 32: 419-425
  6. Gopal, G.J., Kumar, A., Pal, J. and Mukhopadhyay, G. (2014). Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system. Bioengineered. 5(2): 107-113.
  7. Grynberg, M., Li, Z., Szczurek, E. and Godzik, A. (2007). Putative type IV secretion genes in Bacillus anthracis. Trends Microbiol. 15(5): 191-195.http://www.cbs.dtu.dk/services/TMHMM/
  8. Kumar, A., Misra, P., Sisodia, B., Shasany, A.K., Sundar, S. and Dube, A. (2013). Mass spectrometry based proteomic analysis of L. donovani soluble proteins in Indian clinical isolate. Pathog. Dis. 70(1): 4-87. 
  9. Kumar, A., Samant, M., Misra, P., Khare, P., Sundar, S. and Dube, A. (2015). Immunostimulatory potentialand proteome profiling of Leishmaniadonovani soluble exogenous antigens. Parasite Immunol. 37(7): 368-375.
  10. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685.
  11. Okinaka, R.T., Cloud, K., Hampton, O., Hoffmaster, A.R., Hill, K.K., Keim, P., Koehler, T.M., Lamke, G., Kumano, S., Mahillon, J., Manter, D., Martinez, Y., Ricke, D. and Svensson, R. (1999). Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J. Bacteriol. 181(20): 6509-6515.
  12. Rajput, M., Kamboh, A.A., Dewani, P., Umrani, A.P., Abro S.H. and Khan M.A. (2018). Prevalence of Bacillus anthracis spores in wool, hairs and habitat of small ruminants. Indian J. Animal Res. (52):131-135
  13. Redzej, A., Ukleja, M., Connery, S., Trokter, M., Felisberto-Rodrigues, C., Cryar, A., Thalassinos, K., Hayward, R.D., Orlova, E.V. and Waksman, G. (2017). Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery. The EMBO J. 36: 080-3095.
  14. Rivera, J., Cordero, R.J., Nakouzi, A.S., Frases, S., Nicola, A. and Casadevall, A. (2020). Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins. Proc. Natl. Acad. Sci. USA 107:19002-19007.
  15. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. 2nd ed.
  16. Sivasakthi, R., Kumar, T.S., Padmanath, K., Chandrasekar, M., Sriram, P. and Pandiyan, V. (2019). Urinary cystatin C as biomarker for identification of kidney disease in dogs. Indian J Animal Res. 53(2): 1-4.
  17. Vasiliki, E.F., Michael. K. and Nicholas, M.G. (2008). Determination of protein oligomerization state: Two approaches based on glutaraldehyde crosslinking. Analyt. Biochem. 373: 404-406.
  18. Wallden, K., Rivera-Calzada, A. and Waksman, G. (2020). Type IV secretion systems: versatility and diversity in function. Cell Microbiol. 12(9): 1203-1212.
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Indian Journal of Animal Research
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    Research Article

    volume 54 issue 12 (december 2020) : 1525-1531,   Doi: 10.18805/ijar.B-3865

    Characterization of Putative Type IV Secretion System of Bacillus anthracis

    G
    Gopal Jee Gopal
    A
    Awanish Kumar
    1C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli-394 350, Surat, Gujarat, India.
    Cite article:- Gopal Jee Gopal, Kumar Awanish (2020). Characterization of Putative Type IV Secretion System of Bacillus anthracis. Indian Journal of Animal Research. 54(12): 1525-1531. doi: 10.18805/ijar.B-3865.

    ABSTRACT

    Background: Bacillus anthracis causes anthrax disease in livestock and occasionally in humans mainly through three toxic proteins: protective antigen, lethal factor and edema factor. Synthesis of the toxic proteins in bacteria and its pathogenic role in the human host is well known. But, how these toxins are secreted from bacterium to the host or in the culture medium is not known. 
    Methods: A group of researchers have shown (through in silico prediction) that several genes of B. anthracis have similarity with the genes which codes the subunit of the Type IV Secretion System (T4SS) known in other bacteria. In this study, we have tried to explore the existence of T4SS in B. anthracis using molecular biology tools. 
    Result: As an initial lead, three genes (GBAA-pXO1-0064, GBAA-pXO1-0085VirB11 and Bas3783) of B. anthracis T4SS (out of more than 10) were cloned and expressed in E. coli. Among them, gene Bas3783 (VirD4 homolog) is genome encoded therefore; it is sequenced and further characterized for its DNA binding activity and oligomerization. Results obtained from this study indicated the existence of T4SS in B. anthracis.

    REFERENCES

    1. Batra, K., Nanda, T., Kumar, A., Kumar, V., Gopal G.J. and Maan, S. (2019). Molecular cloning and expression kinetics of serum interferon stimulated gene for early pregnancy detection. Indian J. Animal Res. (53): 1129-1134.
    2. Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M. and van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495-503.
    3. Fronzes, R., Schafer, E., Wang, L., Saibil, H.R., Orlova, E.V. and Waksman, G. (2009). Structure of a type IV secretion system core complex. Science. 323: 266- 268.
    4. Garufi, G., Butler, E., Missiakas, D. (2008). ESAT-6-like protein secretion in Bacillus anthracis. J. Bacteriol. 190(21): 7004-7011.
    5. Gopal, G.J. and Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. Protein. J. 32: 419-425
    6. Gopal, G.J., Kumar, A., Pal, J. and Mukhopadhyay, G. (2014). Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system. Bioengineered. 5(2): 107-113.
    7. Grynberg, M., Li, Z., Szczurek, E. and Godzik, A. (2007). Putative type IV secretion genes in Bacillus anthracis. Trends Microbiol. 15(5): 191-195.http://www.cbs.dtu.dk/services/TMHMM/
    8. Kumar, A., Misra, P., Sisodia, B., Shasany, A.K., Sundar, S. and Dube, A. (2013). Mass spectrometry based proteomic analysis of L. donovani soluble proteins in Indian clinical isolate. Pathog. Dis. 70(1): 4-87. 
    9. Kumar, A., Samant, M., Misra, P., Khare, P., Sundar, S. and Dube, A. (2015). Immunostimulatory potentialand proteome profiling of Leishmaniadonovani soluble exogenous antigens. Parasite Immunol. 37(7): 368-375.
    10. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685.
    11. Okinaka, R.T., Cloud, K., Hampton, O., Hoffmaster, A.R., Hill, K.K., Keim, P., Koehler, T.M., Lamke, G., Kumano, S., Mahillon, J., Manter, D., Martinez, Y., Ricke, D. and Svensson, R. (1999). Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J. Bacteriol. 181(20): 6509-6515.
    12. Rajput, M., Kamboh, A.A., Dewani, P., Umrani, A.P., Abro S.H. and Khan M.A. (2018). Prevalence of Bacillus anthracis spores in wool, hairs and habitat of small ruminants. Indian J. Animal Res. (52):131-135
    13. Redzej, A., Ukleja, M., Connery, S., Trokter, M., Felisberto-Rodrigues, C., Cryar, A., Thalassinos, K., Hayward, R.D., Orlova, E.V. and Waksman, G. (2017). Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery. The EMBO J. 36: 080-3095.
    14. Rivera, J., Cordero, R.J., Nakouzi, A.S., Frases, S., Nicola, A. and Casadevall, A. (2020). Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins. Proc. Natl. Acad. Sci. USA 107:19002-19007.
    15. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. 2nd ed.
    16. Sivasakthi, R., Kumar, T.S., Padmanath, K., Chandrasekar, M., Sriram, P. and Pandiyan, V. (2019). Urinary cystatin C as biomarker for identification of kidney disease in dogs. Indian J Animal Res. 53(2): 1-4.
    17. Vasiliki, E.F., Michael. K. and Nicholas, M.G. (2008). Determination of protein oligomerization state: Two approaches based on glutaraldehyde crosslinking. Analyt. Biochem. 373: 404-406.
    18. Wallden, K., Rivera-Calzada, A. and Waksman, G. (2020). Type IV secretion systems: versatility and diversity in function. Cell Microbiol. 12(9): 1203-1212.
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