1887

Abstract

is the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in , important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identified genes coding for proteins possibly involved with suppression of ROS-mediated damage, one () coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing of extracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.030643-0
2009-10-01
2020-01-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/10/3411.html?itemId=/content/journal/micro/10.1099/mic.0.030643-0&mimeType=html&fmt=ahah

References

  1. Angelaccio, S. & Bonaccorsi di Patti, M. C. ( 2002; ). Site-directed mutagenesis by the megaprimer PCR method: variations on a theme for simultaneous introduction of multiple mutations. Anal Biochem 306, 346–349.[CrossRef]
    [Google Scholar]
  2. Bischof, D. F., Janis, C., Vilei, E. M., Bertoni, G. & Frey, J. ( 2008; ). Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells. Infect Immun 76, 263–269.[CrossRef]
    [Google Scholar]
  3. Bischof, D. F., Vilei, E. M. & Frey, J. ( 2009; ). Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant. Vet Res 40, 35 [CrossRef]
    [Google Scholar]
  4. Bryk, R., Griffin, P. & Nathan, C. ( 2000; ). Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature 407, 211–215.[CrossRef]
    [Google Scholar]
  5. Caruso, J. P. & Ross, R. F. ( 1990; ). Effects of Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine. Am J Vet Res 51, 227–231.
    [Google Scholar]
  6. Castro, H., Budde, H., Flohé, L., Hofmann, B., Lünsdorf, H., Wissing, J. & Tomás, A. M. ( 2002; ). Specificity and kinetics of a mitochondrial peroxiredoxin of Leishmania infantum. Free Radic Biol Med 33, 1563–1574.[CrossRef]
    [Google Scholar]
  7. Chen, J. R., Weng, C. N., Ho, T. Y., Cheng, I. C. & Lai, S. S. ( 2000; ). Identification of the copper-zinc superoxide dismutase activity in Mycoplasma hyopneumoniae. Vet Microbiol 73, 301–310.[CrossRef]
    [Google Scholar]
  8. Cho, J. G., Dee, S. A., Deen, J., Guedes, A., Trincado, C., Fano, E., Jiang, Y., Faaberg, K., Collins, J. E. & other authors ( 2006; ). Evaluation of the effects of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs. Am J Vet Res 67, 489–493.[CrossRef]
    [Google Scholar]
  9. Choi, J., Choi, S., Choi, J., Cha, M. K., Kim, I. H. & Shin, W. ( 2003; ). Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins. J Biol Chem 278, 49478–49486.[CrossRef]
    [Google Scholar]
  10. Ciprian, A., Pijoan, C., Cruz, T., Camacho, J., Tortora, J., Colmenares, G., Lopez-Revilla, R. & de la Garza, M. ( 1988; ). Mycoplasma hyopneumoniae increases the susceptibility of pigs to experimental Pasteurella multocida pneumonia. Can J Vet Res 52, 434–438.
    [Google Scholar]
  11. DeBey, M. C. & Ross, R. F. ( 1994; ). Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures. Infect Immun 62, 5312–5318.
    [Google Scholar]
  12. du Manoir, J. M., Albright, B. N., Stevenson, G., Thompson, S. H., Mitchell, G. B., Clark, M. E. & Caswell, J. L. ( 2002; ). Variability of neutrophil and pulmonary alveolar macrophage function in swine. Vet Immunol Immunopathol 89, 175–186.[CrossRef]
    [Google Scholar]
  13. Hames, C., Halbedel, S., Hoppert, M., Frey, J. & Stulke, J. ( 2009; ). Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae. J Bacteriol 191, 747–753.[CrossRef]
    [Google Scholar]
  14. Knoops, B., Loumaye, E. & Van Der Eecken, V. ( 2007; ). Evolution of the peroxiredoxins. Subcell Biochem 44, 27–40.
    [Google Scholar]
  15. Li, J., Zhang, W. B., Loukas, A., Lin, R. Y., Ito, A., Zhang, L. H., Jones, M. & McManus, D. P. ( 2004; ). Functional expression and characterization of Echinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage. Gene 326, 157–165.[CrossRef]
    [Google Scholar]
  16. Li, S., Peterson, N. A., Kim, M. Y., Kim, C. Y., Hung, L. W., Yu, M., Lekin, T., Segelke, B. W., Lott, J. S. & Baker, E. N. ( 2005; ). Crystal structure of AhpE from Mycobacterium tuberculosis, a 1-Cys peroxiredoxin. J Mol Biol 346, 1035–1046.[CrossRef]
    [Google Scholar]
  17. Link, A. J., Robison, K. & Church, G. M. ( 1997; ). Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 18, 1259–1313.[CrossRef]
    [Google Scholar]
  18. Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M. & Haesebrouck, F. ( 2008; ). Control of Mycoplasma hyopneumoniae infections in pigs. Vet Microbiol 126, 297–309.[CrossRef]
    [Google Scholar]
  19. Mare, C. J. & Switzer, W. P. ( 1965; ). New species: Mycoplasma hyopneumoniae; a causative agent of virus pig pneumonia. Vet Med Small Anim Clin 60, 841–846.
    [Google Scholar]
  20. Messier, S., Ross, R. F. & Paul, P. S. ( 1990; ). Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae. Am J Vet Res 51, 52–58.
    [Google Scholar]
  21. Minion, F. C., Lefkowitz, E. J., Madsen, M. L., Cleary, B. J., Swartzell, S. M. & Mahairas, G. G. ( 2004; ). The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 186, 7123–7133.[CrossRef]
    [Google Scholar]
  22. Monteiro, G., Horta, B. B., Pimenta, D. C., Augusto, O. & Netto, L. E. ( 2007a; ). Reduction of 1-Cys peroxiredoxins by ascorbate changes the thiol-specific antioxidant paradigm, revealing another function of vitamin C. Proc Natl Acad Sci U S A 104, 4886–4891.[CrossRef]
    [Google Scholar]
  23. Monteiro, K. M., Scapin, S. M., Navarro, M. V., Zanchin, N. I., Cardoso, M. B., da Silveira, N. P., Goncalves, P. F., Stassen, H. K., Zaha, A. & Ferreira, H. B. ( 2007b; ). Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers. Biochim Biophys Acta 1774, 278–285.[CrossRef]
    [Google Scholar]
  24. Ohnishi, Y., Ishikawa, J., Hara, H., Suzuki, H., Ikenoya, M., Ikeda, H., Yamashita, A., Hattori, M. & Horinouchi, S. ( 2008; ). Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 190, 4050–4060.[CrossRef]
    [Google Scholar]
  25. Oshima, K. & Nishida, H. ( 2007; ). Phylogenetic relationships among mycoplasmas based on the whole genomic information. J Mol Evol 65, 249–258.[CrossRef]
    [Google Scholar]
  26. Pilo, P., Vilei, E. M., Peterhans, E., Bonvin-Klotz, L., Stoffel, M. H., Dobbelaere, D. & Frey, J. ( 2005; ). A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony. J Bacteriol 187, 6824–6831.[CrossRef]
    [Google Scholar]
  27. Pinto, P. M., Chemale, G., de Castro, L. A., Costa, A. P., Kich, J. D., Vainstein, M. H., Zaha, A. & Ferreira, H. B. ( 2007; ). Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins. Vet Microbiol 121, 83–93.[CrossRef]
    [Google Scholar]
  28. Pushpamali, W. A., de Zoysa, M., Kang, H. S., Oh, C. H., Whang, I., Kim, S. J. & Lee, J. ( 2008; ). Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis. Fish Shellfish Immunol 24, 294–307.[CrossRef]
    [Google Scholar]
  29. Razin, S., Yogev, D. & Naot, Y. ( 1998; ). Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62, 1094–1156.
    [Google Scholar]
  30. Rhee, S. G., Chae, H. Z. & Kim, K. ( 2005; ). Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 38, 1543–1552.[CrossRef]
    [Google Scholar]
  31. Rho, B. S., Hung, L. W., Holton, J. M., Vigil, D., Kim, S. I., Park, M. S., Terwilliger, T. C. & Pedelacq, J. D. ( 2006; ). Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis. J Mol Biol 361, 850–863.[CrossRef]
    [Google Scholar]
  32. Ross, R. F. ( 1992; ). Mycoplasmal disease. In Diseases of Swine, pp. 535–549. Edited by B. E. Straw. Ames, IA: Iowa State University Press.
  33. Schafer, E. R., Oneal, M. J., Madsen, M. L. & Minion, F. C. ( 2007; ). Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to hydrogen peroxide. Microbiology 153, 3785–3790.[CrossRef]
    [Google Scholar]
  34. Seo, M. S., Kang, S. W., Kim, K., Baines, I. C., Lee, T. H. & Rhee, S. G. ( 2000; ). Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. J Biol Chem 275, 20346–20354.[CrossRef]
    [Google Scholar]
  35. Sirand-Pugnet, P., Citti, C., Barre, A. & Blanchard, A. ( 2007; ). Evolution of mollicutes: down a bumpy road with twists and turns. Res Microbiol 158, 754–766.[CrossRef]
    [Google Scholar]
  36. Stinear, T. P., Seemann, T., Pidot, S., Frigui, W., Reysset, G., Garnier, T., Meurice, G., Simon, D., Bouchier, C. & other authors ( 2007; ). Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res 17, 192–200.[CrossRef]
    [Google Scholar]
  37. Suttiprapa, S., Loukas, A., Laha, T., Wongkham, S., Kaewkes, S., Gaze, S., Brindley, P. J. & Sripa, B. ( 2008; ). Characterization of the antioxidant enzyme, thioredoxin peroxidase, from the carcinogenic human liver fluke, Opisthorchis viverrini. Mol Biochem Parasitol 160, 116–122.[CrossRef]
    [Google Scholar]
  38. Tamura, K., Dudley, J., Nei, M. & Kumar, S. ( 2007; ). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599.[CrossRef]
    [Google Scholar]
  39. Vasconcelos, A. T., Ferreira, H. B., Bizarro, C. V., Bonatto, S. L., Carvalho, M. O., Pinto, P. M., Almeida, D. F., Almeida, L. G., Almeida, R. & other authors ( 2005; ). Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J Bacteriol 187, 5568–5577.[CrossRef]
    [Google Scholar]
  40. Vilei, E. M. & Frey, J. ( 2001; ). Genetic and biochemical characterization of glycerol uptake in Mycoplasma mycoides subsp. mycoides SC: its impact on H2O2 production and virulence. Clin Diagn Lab Immunol 8, 85–92.
    [Google Scholar]
  41. Wang, Q., Chen, K., Yao, Q., Zhao, Y., Li, Y., Shen, H. & Mu, R. ( 2008; ). Identification and characterization of a novel 1-Cys peroxiredoxin from silkworm, Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol 149, 176–182.[CrossRef]
    [Google Scholar]
  42. Wood, Z. A., Schroder, E., Robin, H. J. & Poole, L. B. ( 2003; ). Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28, 32–40.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.030643-0
Loading
/content/journal/micro/10.1099/mic.0.030643-0
Loading

Data & Media loading...

Supplements

MhPrx peroxidase activity assay dataset used in the ANOVA and in the Tukey's multiple comparison tests [ PDF] (14 kb) Detection of MhPrx expression in protein extracts from different strains [ PDF] (190 kb) Multiple sequence alignment of MhPrx and bacterial members of the 1-Cys peroxiredoxin subfamily [ PDF] (166 kb)

PDF

MhPrx peroxidase activity assay dataset used in the ANOVA and in the Tukey's multiple comparison tests [ PDF] (14 kb) Detection of MhPrx expression in protein extracts from different strains [ PDF] (190 kb) Multiple sequence alignment of MhPrx and bacterial members of the 1-Cys peroxiredoxin subfamily [ PDF] (166 kb)

PDF

MhPrx peroxidase activity assay dataset used in the ANOVA and in the Tukey's multiple comparison tests [ PDF] (14 kb) Detection of MhPrx expression in protein extracts from different strains [ PDF] (190 kb) Multiple sequence alignment of MhPrx and bacterial members of the 1-Cys peroxiredoxin subfamily [ PDF] (166 kb)

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error