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  • 1 Qatar University, Doha, State of Qatar
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L. pneumophila is an intracellular bacterium that replicates inside a membrane-bound vacuole called Legionella-containing vacuole (LCV), where it plentifully liberates its HtpB chaperonin. From LCV, HtpB reaches the host cell cytoplasm, where it interacts with SAMDC, a cytoplasmic protein required for synthesis of host polyamines that are important for intracellular growth of L. pneumophila. Additionally, cytoplasmic expression of HtpB in S. cerevisiae induces pseudohyphal growth, and in mammalian cells recruits mitochondria to LCV, and modifies actin microfilaments organization. This led us to hypothesize here that HtpB recruits a protein(s) from eukaryotic cells that is involved in the emergence of the aforementioned phenotypes. To identify this protein, a commercially available HeLa cDNA library was screened using a yeast two-hybrid system. Approximately 5×106 yeast clones carrying HeLa cDNA library plasmid were screened. Twenty-one positive clones were identified. DNA sequence analysis revealed that all of these positive clones encoded the mammalian small heat shock protein Hsp10. Based on the fact that chaperonions are required to interact with co-chaperonins to function properly in protein folding, we believe that HtpB recruits the host cell Hsp10 to appropriately interact with SAMDC and to induce the multifunction phenotypes deemed important in L. pneumophila pathogenesis.

  • 1.

    Fernandez-Moreira, E., Helbig, J.H., Swanson, M.S.: Membrane vesicles shed by Legionella pneumophila inhibit fusion of phagosomes with lysosomes. Infect Immun 74, 32853295 (2006).

    • Search Google Scholar
    • Export Citation
  • 2.

    Fernandez, R.C., Logan, S.M., Lee, S.H., Hoffman, P.S.: Elevated levels of Legionella pneumophila stress protein Hsp60 early in infection of human monocytes and L929 cells correlate with virulence. Infect Immun 64, 19681976 (1996).

    • Search Google Scholar
    • Export Citation
  • 3.

    Garduno, R.A., Chong, A., Nasrallah, G.K., Allan, D.S.: The Legionella pneumophila Chaperonin — An Unusual Multifunctional Protein in Unusual Locations. Front Microbiol 2, 122 (2011).

    • Search Google Scholar
    • Export Citation
  • 4.

    Gupta, R.S.: Evolution of the chaperonin families (Hsp60, Hsp10 and Tcp-1) of proteins and the origin of eukaryotic cells. Mol Microbiol 15, 111 (1995).

    • Search Google Scholar
    • Export Citation
  • 5.

    Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C.: The universally conserved GroE (Hsp60) chaperonins. Annu Rev Microbiol 45, 301325 (1991).

    • Search Google Scholar
    • Export Citation
  • 6.

    Nasrallah, G.K., Gagnon, E., Orton, D.J., Garduno, R.A.: The htpAB operon of Legionella pneumophila cannot be deleted in the presence of the groE chaperonin operon of Escherichia coli. Can J Microbiol 57, 943952 (2011).

    • Search Google Scholar
    • Export Citation
  • 7.

    Nasrallah, G.K., Riveroll, A.L., Chong, A., Murray, L.E., Lewis, P.J., Garduno, R.A.: Legionella pneumophila requires polyamines for optimal intracellular growth. J Bacteriol 193, 43464360 (2011).

    • Search Google Scholar
    • Export Citation
  • 8.

    Bonshtien, A.L., Parnas, A., Sharkia, R., Niv, A., Mizrahi, Azem A, Weiss, C.: Differential effects of co-chaperonin homologs on cpn60 oligomers. Cell Stress Chaperones 14, 509519 (2009).

    • Search Google Scholar
    • Export Citation
  • 9.

    Hoffman, P.S., Houston, L., Butler, C.A.: Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila infected HeLa cells. Infect Immun 58, 33803387 (1990).

    • Search Google Scholar
    • Export Citation
  • 10.

    Hoffman, P.S., Seyer, J.H., Butler, C.A.: Molecular characterization of the 28-and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein. J Bacteriol 174, 908913 (1992).

    • Search Google Scholar
    • Export Citation
  • 11.

    Czarnecka, A.M., Campanella, C., Zummo, G., Cappello, F.: Heat shock protein 10 and signal transduction: a “capsulaeburnea” of carcinogenesis? Cell Stress Chaperones 11, 287294 (2006).

    • Search Google Scholar
    • Export Citation
  • 12.

    Hartman, D.J., Hoogenraad, N.J., Condron, R., Hoj, P.B.: Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric ornithine transcarbamoylase in vitro. Proc Natl Acad Sci USA 89, 33943398 (1992).

    • Search Google Scholar
    • Export Citation
  • 13.

    Garduno, R.A., Faulkner, G., Trevors, M.A., Vats, N., Hoffman, P.S.: Immunolocalization of Hsp60 in Legionella pneumophila. J Bacteriol 180, 505513 (1998).

    • Search Google Scholar
    • Export Citation
  • 14.

    Blander, S.J., Horwitz, M.A.: Major cytoplasmic membrane protein of Legionella pneumophila, a genus common antigen and member of the Hsp 60 family of heat shock proteins, induces protective immunity in a guinea pig model of Legionnaires’ disease. J Clin Invest 91, 717723 (1993).

    • Search Google Scholar
    • Export Citation
  • 15.

    Gabay, J.E, Horwitz, M.A.: Isolation and characterization of the cytoplasmic and outer membranes of the Legionnaires’ disease bacterium (Legionella pneumophila). J Exp Med 161, 409422 (1985).

    • Search Google Scholar
    • Export Citation
  • 16.

    Garduno, R.A., Garduno, E., Hoffman, P.S.: Surface-associated Hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model. Infect Immun 66, 46024610 (1998).

    • Search Google Scholar
    • Export Citation
  • 17.

    Garduno, R.A., Garduno, E., Hiltz, M., Hoffman, P.S.: Intracellular growth of Legionella pneumophila gives rise to a differentiated form dissimilar to stationary-phase forms. Infect Immun 70, 62736283 (2002).

    • Search Google Scholar
    • Export Citation
  • 18.

    Chong, A., Lima, C.A., Allan, D.S., Nasrallah, G.K., Garduno, R.A.: The purified and recombinant Legionella pneumophila chaperonin alters mitochondrial trafficking and microfilament organization. Infect Immun 77, 47244739 (2009).

    • Search Google Scholar
    • Export Citation
  • 19.

    Guthrie, C., Fink, G.R.: Guide to yeast genetics and molecular biology. Methods Enzymol 194, 1863 (1991).

  • 20.

    Clontech, L.I.: Matchmaker GAL4 two-hybrid system 3 & libraries user manual. 38 (1999).

  • 21.

    Gietz, D., St, J.A., Woods, R.A., Schiestl, R.H.: Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20, 1425 (1992).

    • Search Google Scholar
    • Export Citation
  • 22.

    Nasrallah, G.K.: Potential roles of the chaperonin (HtpB), polyamines, and the polyamine binding protein (PotD) in Legionella pneumophila pathogenesis. Ph.D Thesis. 2011. Published online (Dal space). Dalhousie University, Nova Scotia, Canada.

    • Search Google Scholar
    • Export Citation
  • 23.

    Towbin, H., Staehelin, T., Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24, 145149 (1979).

    • Search Google Scholar
    • Export Citation
  • 24.

    Riveroll, A.: The Legionella pneumophila chaperonin — An investigation of virulencerelated roles in a yeast model. Ph.D thesis. 2005. Dalhousie University, NS, Canada.

    • Search Google Scholar
    • Export Citation
  • 25.

    Sadacharan, S.K., Cavanagh, A.C., Gupta, R.S.: Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules. Histochem Cell Biol 116, 507517 (2001).

    • Search Google Scholar
    • Export Citation
  • 26.

    Fields, S.: High-throughput two-hybrid analysis. The promise and the peril. FEBS 272, 53915399 (2005).

  • 27.

    Fields, S.: Interactive learning: lessons from two hybrids over two decades. Proteomics 9, 52095213 (2009).

  • 28.

    Pegg, A.E., Xiong, H., Feith, D.J., Shantz, L.M.: S-adenosylmethionine decarboxylase: structure, function and regulation by polyamines. Biochem Soc Trans 26, 580586 (1998).

    • Search Google Scholar
    • Export Citation
  • 29.

    Pegg, A.E.: S-Adenosylmethionine decarboxylase. Essays Biochem 46, 2545 (2009).

  • 30.

    Pegg, A.E.: Recent advances in the biochemistry of polyamines in eukaryotes. Biochem 234, 249262 (1986).

  • 31.

    Kashiwagi, K., Taneja, S.K., Liu, T.Y., Tabor, C.W., Tabor, H.: Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase. J Biol Chem 265, 2232122328 (1990).

    • Search Google Scholar
    • Export Citation
  • 32.

    Pegg, A.E., Kameji, T., Shirahata, A., Stanley, B., Madhubala, R., Pajunen, A.: Regulation of mammalian S-adenosylmethionine decarboxylase. Adv Enzyme Regul 27, 703714 (1988).

    • Search Google Scholar
    • Export Citation
  • 33.

    Nasrallah, G.K., Abdelhady, H., Tompkins N.P., Carson, K.R., Garduño, R.A.: Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila. Int J Med Microbiol 304, 703714 (2014).

    • Search Google Scholar
    • Export Citation

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