1887

Abstract

Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease. PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors. The metalloprotease also shows a calcium- and temperature-dependent autolysis. The gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from , and . The gene resides in a locus encoding both the protease ABC transporter () and an intervening ORF encoding a protease inhibitor (). PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system. Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.

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2003-06-01
2019-10-19
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References

  1. Ahn, J. H., Pan, J. G. & Rhee, J. S. ( 1999; ). Identification of the TliDEFABC transporter specific for lipase in Pseudomonas fluorescens SIK W1. J Bacteriol 181, 1847–1852.
    [Google Scholar]
  2. Akatsuka, H., Kawai, E., Omori, K. & Shibatani, T. ( 1995; ). The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide. J Bacteriol 177, 6381–6389.
    [Google Scholar]
  3. Akatsuka, H., Binet, R., Kawai, E., Wandersman, C. & Omori, K. ( 1997; ). Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters. J Bacteriol 179, 4754–4760.
    [Google Scholar]
  4. Bowen, D., Rocheleau, T. A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R. & ffrench-Constant, R. H. ( 1998; ). Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129–2132.[CrossRef]
    [Google Scholar]
  5. Bowen, D., Blackburn, M., Rocheleau, T., Grutzmacher, C. & ffrench-Constant, R. H. ( 2000; ). Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes. Insect Biochem Mol Biol 30, 69–74.[CrossRef]
    [Google Scholar]
  6. Caldas, C., Cherqui, A., Pereira, A. & Simoes, N. ( 2002; ). Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression. Appl Environ Microbiol 68, 1297–1304.[CrossRef]
    [Google Scholar]
  7. Daborn, P. J., Waterfield, N., Blight, M. A. & ffrench-Constant, R. H. ( 2001; ). Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection. J Bacteriol 183, 5834–5839.[CrossRef]
    [Google Scholar]
  8. Dahler, G. S., Barras, F. & Keen, N. T. ( 1990; ). Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16. J Bacteriol 172, 5803–5815.
    [Google Scholar]
  9. Delepelaire, P. & Wandersman, C. ( 1990; ). Protein secretion in gram-negative bacteria. J Biol Chem 265, 17118–17125.
    [Google Scholar]
  10. Duong, F., Lazdunski, A., Cami, B. & Murgier, M. ( 1992; ). Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways. Gene 121, 47–54.[CrossRef]
    [Google Scholar]
  11. Everett, M., Walsh, T., Guay, G. & Bennett, P. ( 1995; ). GcvA, a LysR-type transcriptional regulator protein, activates expression of the cloned Citrobacter freundii ampC β-lactamase gene in Escherichia coli: cross-talk between DNA-binding proteins. Microbiology 141, 419–430.[CrossRef]
    [Google Scholar]
  12. ffrench-Constant, R., Waterfield, N., Daborn, P. & 7 other authors ( 2003; ). Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen. FEMS Microbiol Rev 26, 433–456.[CrossRef]
    [Google Scholar]
  13. Fischer-Le Saux, M., Viallard, V., Brunel, B., Normand, P. & Boemare, N. E. ( 1999; ). Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov. Int J Syst Bacteriol 49, 1645–1656.[CrossRef]
    [Google Scholar]
  14. Forst, S. & Clarke, D. ( 2001; ). Bacteria-nematode symbiosis. In Entomopathogenic Nematology, pp. 57–77. Edited by R. Gaugler. London: CABI.
  15. Forst, S., Dowds, B., Boemare, N. & Stackebrandt, E. ( 1997; ). Xenorhabdus and Photorhabdus spp. bugs that kill bugs. Annu Rev Microbiol 51, 47–72.[CrossRef]
    [Google Scholar]
  16. Ghigo, J. M. & Wandersman, C. ( 1992a; ). Cloning, nucleotide sequence and characterization of the gene encoding the Erwinia chrysanthemi B374 PrtA metalloprotease: a third metalloprotease secreted via a C-terminal secretion signal. Mol Gen Genet 236, 135–144.
    [Google Scholar]
  17. Ghigo, J. M. & Wandersman, C. ( 1992b; ). A fourth metalloprotease gene in Erwinia chrysanthemi. Res Microbiol 143, 857–867.[CrossRef]
    [Google Scholar]
  18. Guzzo, J., Murgier, M., Filloux, A. & Lazdunski, A. ( 1990; ). Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli. J Bacteriol 172, 942–948.
    [Google Scholar]
  19. Guzzo, J., Duong, F., Wandersman, C., Murgier, M. & Lazdunski, A. ( 1991a; ). The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli α-haemolysin. Mol Microbiol 5, 447–453.[CrossRef]
    [Google Scholar]
  20. Guzzo, J., Pages, J. M., Duong, F., Lazdunski, A. & Murgier, M. ( 1991b; ). Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism. J Bacteriol 173, 5290–5297.
    [Google Scholar]
  21. Hammond, S. E. & Hanna, P. C. ( 1998; ). Lethal factor active-site mutations affect catalytic activity in vitro. Infect Immun 66, 2374–2378.
    [Google Scholar]
  22. Hanna, P. ( 1999; ). Lethal toxin actions and their consequences. J Appl Microbiol 87, 285–287.[CrossRef]
    [Google Scholar]
  23. Jarosz, J., Balcerzak, M. & Skrzypek, H. ( 1991; ). Involvement of larvicidal toxin in pathogenesis of insect parasitism with the Rhabditoid nematodes Steinernema feltiae and Heterorhabditis bacteriophora. Entomophaga 36, 361–368.[CrossRef]
    [Google Scholar]
  24. Kawai, E., Idei, A., Kumura, H., Shimazaki, K., Akatsuka, H. & Omori, K. ( 1999; ).The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33. Biochim Biophys Acta 1446, 377–382.[CrossRef]
    [Google Scholar]
  25. Kling, J. J., Wright, R. L., Moncrief, J. S. & Wilkins, T. D. ( 1997; ). Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis. FEMS Microbiol Lett 146, 279–284.[CrossRef]
    [Google Scholar]
  26. Letoffe, S., Delepelaire, P. & Wandersman, C. ( 1990; ). Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli α-haemolysin. EMBO J 9, 1375–1382.
    [Google Scholar]
  27. Letoffe, S., Delepelaire, P. & Wandersman, C. ( 1991; ). Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions. J Bacteriol 173, 2160–2166.
    [Google Scholar]
  28. Miyoshi, S. & Shinoda, S. ( 2000; ). Microbial metalloproteases and pathogenesis. Microbes Infect 2, 91–98.[CrossRef]
    [Google Scholar]
  29. Moncrief, J. S., Obiso, R., Jr, Barroso, L. A., Kling, J. J., Wright, R. L., Van Tassell, R. L., Lyerly, D. M. & Wilkins, T. D. ( 1995; ). The enterotoxin of Bacteroides fragilis is a metalloprotease. Infect Immun 63, 175–181.
    [Google Scholar]
  30. Nakahama, K., Yoshimura, K., Marumoto, R., Kikuchi, M., Lee, I. S., Hase, T. & Matsubara, H. ( 1986; ). Cloning and sequencing of Serratia protease gene. Nucleic Acids Res 14, 5843–5855.[CrossRef]
    [Google Scholar]
  31. Olson, J. C. & Ohman, D. E. ( 1992; ). Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions. J Bacteriol 174, 4140–4147.
    [Google Scholar]
  32. Ong, K. L. & Chang, F. N. ( 1997; ). Analysis of proteins from different phase variants of the entomopathogenic bacteria Photorhabdus luminescens by two-dimensional zymography. Electrophoresis 18, 834–839.[CrossRef]
    [Google Scholar]
  33. Raveneau, J., Geoffroy, C., Beretti, J. L., Gaillard, J. L., Alouf, J. E. & Berche, P. ( 1992; ). Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene. Infect Immun 60, 916–921.
    [Google Scholar]
  34. Schmidt, T. M., Bleakley, B. & Nealson, K. H. ( 1988; ). Characterization of an extracellular protease from the insect pathogen Xenorhabdus luminescens. Appl Environ Microbiol 54, 2793–2797.
    [Google Scholar]
  35. Silva, C. P., Waterfield, N. R., Daborn, P. J. & 7 other authors ( 2002; ). Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell Microbiol 6, 329–339.
    [Google Scholar]
  36. Spencker, F. B., Haupt, S., Claros, M. C., Walter, S., Lietz, T., Schille, R. & Rodloff, A. C. ( 2000; ). Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis. Clin Microbiol Infect 6, 600–607.[CrossRef]
    [Google Scholar]
  37. Tonello, F., Morante, S., Rossetto, O., Schiavo, G. & Montecucco, C. ( 1996; ). Tetanus and botulism neurotoxins: a novel group of zinc-endopeptidases. Adv Exp Med Biol 389, 251–260.
    [Google Scholar]
  38. Valens, M., Broutelle, A. C., Lefebvre, M. & Blight, M. A. ( 2002; ). A zinc metalloprotease inhibitor, Inh, from the insect pathogen, Photorhabdus luminescens. Microbiology 148, 2427–2437.
    [Google Scholar]
  39. Wang, H. & Dowds, B. C. ( 1993; ). Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium. J Bacteriol 175, 1665–1673.
    [Google Scholar]
  40. Waterfield, N., Dowling, A., Sharma, S., Daborn, P. J., Potter, U. & ffrench-Constant, R. H. ( 2001; ). Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli. Appl Environ Microbiol 67, 5017–5024.[CrossRef]
    [Google Scholar]
  41. Wee, K. E., Yonan, C. R. & Chang, F. N. ( 2000; ). A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens. Microbiology 146, 3141–3147.
    [Google Scholar]
  42. Welch, R. A. ( 1991; ). Pore-forming cytolysins of gram-negative bacteria. Mol Microbiol 5, 521–528.[CrossRef]
    [Google Scholar]
  43. Yamanaka, S., Hagiwara, A., Nishimura, Y., Tanabe, H. & Ishibashi, N. ( 1992; ). Biochemical and physiological characteristics of Xenorhabdus species, symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae). Arch Microbiol 158, 387–393.
    [Google Scholar]
  44. Yoshihara, K., Matsushita, O., Minami, J. & Okabe, A. ( 1994; ). Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. J Bacteriol 176, 6489–6496.
    [Google Scholar]
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