1887

Abstract

adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (), interacts with host-cell receptor Hsp60 to promote bacterial adhesion during the intestinal phase of infection. The LAP homologue is present in pathogens (, ) and non-pathogens (, , ); however, its role in non-pathogens is unknown. Sequence analysis revealed 98 % amino acid similarity in LAP from all species. The N-terminus contains acetaldehyde dehydrogenase (ALDH) and the C-terminus an alcohol dehydrogenase (ADH). Recombinant LAP from , , and exhibited ALDH and ADH activities, and displayed strong binding affinity ( 2–31 nM) towards Hsp60. Flow cytometry, ELISA and immunoelectron microscopy revealed more surface-associated LAP in pathogens than non-pathogens. Pathogens exhibited significantly higher adhesion (<0.05) to Caco-2 cells than non-pathogens; however, pretreatment of bacteria with Hsp60 caused 47–92 % reduction in adhesion only in pathogens. These data suggest that biochemical properties of LAP from pathogenic are similar to those of the protein from non-pathogens in many respects, such as substrate specificity, immunogenicity, and binding affinity to Hsp60. However, protein fractionation analysis of extracts from pathogenic and non-pathogenic species revealed that LAP was greatly reduced in intracellular and cell-surface protein fractions, and undetectable in the extracellular milieu of non-pathogens even though the transcript levels were similar for both. Furthermore, a LAP preparation from restored adhesion in a mutant (KB208) of but not in , indicating possible lack of surface reassociation of LAP molecules in this bacterium. Taken together, these data suggest that LAP expression level, cell-surface localization, secretion and reassociation are responsible for LAP-mediated pathogenicity and possibly evolved to adapt to a parasitic life cycle in the host.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.036509-0
2010-09-01
2019-08-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/9/2782.html?itemId=/content/journal/micro/10.1099/mic.0.036509-0&mimeType=html&fmt=ahah

References

  1. Autret, N., Raynaud, C., Dubail, I., Berche, P. & Charbit, A. ( 2003; ). Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence. Infect Immun 71, 4463–4471.[CrossRef]
    [Google Scholar]
  2. Bakardjiev, A. I., Theriot, J. A. & Portnoy, D. A. ( 2006; ). Listeria monocytogenes traffics from maternal organs to the placenta and back. PLoS Pathog 2, e66.[CrossRef]
    [Google Scholar]
  3. Bennett, M. R., Pang, W. L., Ostroff, N. A., Baumgartner, B. L., Nayak, S., Tsimring, L. S. & Hasty, J. ( 2008; ). Metabolic gene regulation in a dynamically changing environment. Nature 454, 1119–1122.[CrossRef]
    [Google Scholar]
  4. Bergmann, S., Rohde, M., Chhatwal, G. S. & Hammerschmidt, S. ( 2001; ). α-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol Microbiol 40, 1273–1287.[CrossRef]
    [Google Scholar]
  5. Bhunia, A. K. & Johnson, M. G. ( 1992; ). Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen. Appl Environ Microbiol 58, 1924–1929.
    [Google Scholar]
  6. Bhunia, A. K., Ball, P. H., Fuad, A. T., Kurz, B. W., Emerson, J. W. & Johnson, M. G. ( 1991; ). Development and characterization of a monoclonal antibody specific for Listeria monocytogenes and Listeria innocua. Infect Immun 59, 3176–3184.
    [Google Scholar]
  7. Bierne, H. & Cossart, P. ( 2007; ). Listeria monocytogenes surface proteins: from genome predictions to function. Microbiol Mol Biol Rev 71, 377–397.[CrossRef]
    [Google Scholar]
  8. Blandino, A., Caro, I. & Cantero, D. ( 1997; ). Comparative study of alcohol dehydrogenase activity in flor yeast extracts. Biotechnol Lett 19, 651–654.[CrossRef]
    [Google Scholar]
  9. Braun, L., Dramsi, S., Dehoux, P., Bierne, H., Lindahl, G. & Cossart, P. ( 1997; ). InIB: an invasion protein of Listeria monocytogenes with a novel type of surface association. Mol Microbiol 25, 285–294.[CrossRef]
    [Google Scholar]
  10. Bubert, A., Kuhn, M., Goebel, W. & Kohler, S. ( 1992; ). Structural and functional properties of the p60 proteins from different Listeria species. J Bacteriol 174, 8166–8171.
    [Google Scholar]
  11. Bubert, A., Hein, I., Rauch, M., Lehner, A., Yoon, B., Goebel, W. & Wagner, M. ( 1999; ). Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl Environ Microbiol 65, 4688–4692.
    [Google Scholar]
  12. Bueno, V. F., Banerjee, P., Banada, P. P., de Mesquita, A. J., Lemes-Marques, E. G. & Bhunia, A. K. ( 2010; ). Characterization of Listeria monocytogenes isolates of food and human origins from Brazil using molecular typing procedures and in vitro cell culture assays. Int J Environ Health Res 20, 43–59.[CrossRef]
    [Google Scholar]
  13. Burkholder, K. M., Kim, K.-P., Mishra, K. K., Medina, S., Hahm, B.-K., Kim, H. & Bhunia, A. K. ( 2009; ). Expression of LAP, a SecA2-dependent secretory protein, is induced under anaerobic environment. Microbes Infect 11, 859–867.[CrossRef]
    [Google Scholar]
  14. Chhatwal, G. S. ( 2002; ). Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors. Trends Microbiol 10, 205–208.[CrossRef]
    [Google Scholar]
  15. Drevets, D. A. & Bronze, M. S. ( 2008; ). Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion. FEMS Immunol Med Microbiol 53, 151–165.[CrossRef]
    [Google Scholar]
  16. Espinosa, A., Yan, L., Zhang, Z., Foster, L., Clark, D., Li, E. & Stanley, S. L., Jr ( 2001; ). The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dehydrogenase and acetaldehyde dehydrogenase activity. J Biol Chem 276, 20136–20143.[CrossRef]
    [Google Scholar]
  17. Freitag, N. E., Port, G. C. & Miner, M. D. ( 2009; ). Listeria monocytogenes from saprophyte to intracellular pathogen. Nat Rev Microbiol 7, 623–628.[CrossRef]
    [Google Scholar]
  18. Fujimoto, S. & Ike, Y. ( 2001; ). pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis. Appl Environ Microbiol 67, 1262–1267.[CrossRef]
    [Google Scholar]
  19. Gahan, C. G. & Hill, C. ( 2005; ). Gastrointestinal phase of Listeria monocytogenes infection. J Appl Microbiol 98, 1345–1353.[CrossRef]
    [Google Scholar]
  20. Gaillard, J. L., Berche, P., Frehel, C., Gouin, E. & Cossart, P. ( 1991; ). Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell 65, 1127–1141.[CrossRef]
    [Google Scholar]
  21. Gil-Navarro, I., Gil, M. L., Casanova, M., O'Connor, J. E., Martinez, J. P. & Gozalbo, D. ( 1997; ). The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen. J Bacteriol 179, 4992–4999.
    [Google Scholar]
  22. Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P. & other authors ( 2001; ). Comparative genomics of Listeria species. Science 294, 849–852.
    [Google Scholar]
  23. Gupta, S., Mat-Jan, F., Latifi, M. & Clark, D. P. ( 2000; ). Acetaldehyde dehydrogenase activity of the AdhE protein of Escherichia coli is inhibited by intermediates in ubiquinone synthesis. FEMS Microbiol Lett 182, 51–55.[CrossRef]
    [Google Scholar]
  24. Hamon, M., Bierne, H. & Cossart, P. ( 2006; ). Listeria monocytogenes: a multifaceted model. Nat Rev Microbiol 4, 423–434.[CrossRef]
    [Google Scholar]
  25. Henderson, B., Allan, E. & Coates, A. R. ( 2006; ). Stress wars: the direct role of host and bacterial molecular chaperones in bacterial infection. Infect Immun 74, 3693–3706.[CrossRef]
    [Google Scholar]
  26. Holmes, A. R., McNab, R., Millsap, K. W., Rohde, M., Hammerschmidt, S., Mawdsley, J. L. & Jenkinson, H. F. ( 2001; ). The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. Mol Microbiol 41, 1395–1408.[CrossRef]
    [Google Scholar]
  27. Jacob, F. & Monod, J. ( 1961; ). Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3, 318–356.[CrossRef]
    [Google Scholar]
  28. Jaradat, Z. W., Wampler, J. W. & Bhunia, A. W. ( 2003; ). A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines. Med Microbiol Immunol 192, 85–91.
    [Google Scholar]
  29. Jelski, W., Chrostek, L., Szmitkowski, M. & Markiewicz, W. ( 2006; ). The activity of class I, II, III and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in breast cancer. Clin Exp Med 6, 89–93.[CrossRef]
    [Google Scholar]
  30. Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M. & Cossart, P. ( 2002; ). An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551–561.[CrossRef]
    [Google Scholar]
  31. Jonquieres, R., Bierne, H., Fiedler, F., Gounon, P. & Cossart, P. ( 1999; ). Interaction between the protein InIB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of Gram-positive bacteria. Mol Microbiol 34, 902–914.[CrossRef]
    [Google Scholar]
  32. Kim, K.-P. ( 2004; ). Genetic identification and characterization of Listeria adhesion protein, an alcohol acetaldehyde dehydrogenase homologue in Listeria monocytogenes. PhD thesis, Department of Food Science, Purdue University.
  33. Kim, K. P., Jagadeesan, B., Burkholder, K. M., Jaradat, Z. W., Wampler, J. L., Lathrop, A. A., Morgan, M. T. & Bhunia, A. K. ( 2006; ). Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor. FEMS Microbiol Lett 256, 324–332.[CrossRef]
    [Google Scholar]
  34. Kinhikar, A. G., Vargas, D., Li, H., Mahaffey, S. B., Hinds, L., Belisle, J. T. & Laal, S. ( 2006; ). Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin. Mol Microbiol 60, 999–1013.[CrossRef]
    [Google Scholar]
  35. Koo, O. K., Jeong, D. W., Lee, J. M., Kim, M. J., Lee, J. H., Chang, H. C., Kim, J. H. & Lee, H. J. ( 2005; ). Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Leuconostoc mesenteroides isolated from kimchi. Biotechnol Lett 27, 505–510.[CrossRef]
    [Google Scholar]
  36. Koo, O. K., Liu, Y., Shuaib, S., Bhattacharya, S., Ladisch, M. R., Bashir, R. & Bhunia, A. K. ( 2009; ). Targeted capture of pathogenic bacteria using a mammalian cell receptor coupled with dielectrophoresis on a biochip. Anal Chem 81, 3094–3101.[CrossRef]
    [Google Scholar]
  37. Lathrop, A. A., Jaradat, Z. W., Haley, T. & Bhunia, A. K. ( 2003; ). Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor. J Immunol Methods 281, 119–128.[CrossRef]
    [Google Scholar]
  38. Lecuit, M., Nelson, D. M., Smith, S. D., Khun, H., Huerre, M., Vacher-Lavenu, M. C., Gordon, J. I. & Cossart, P. ( 2004; ). Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin. Proc Natl Acad Sci U S A 101, 6152–6157.[CrossRef]
    [Google Scholar]
  39. Lenz, L. L. & Portnoy, D. A. ( 2002; ). Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype. Mol Microbiol 45, 1043–1056.[CrossRef]
    [Google Scholar]
  40. Lenz, L. L., Mohammadi, S., Geissler, A. & Portnoy, D. A. ( 2003; ). SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc Natl Acad Sci U S A 100, 12432–12437.[CrossRef]
    [Google Scholar]
  41. Machata, S., Hain, T., Rohde, M. & Chakraborty, T. ( 2005; ). Simultaneous deficiency of both MurA and p60 proteins generates a rough phenotype in Listeria monocytogenes. J Bacteriol 187, 8385–8394.[CrossRef]
    [Google Scholar]
  42. Membrillo-Hernandez, J., Echave, P., Cabiscol, E., Tamarit, J., Ros, J. & Lin, E. C. ( 2000; ). Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. Genetic and biochemical studies of the mutant proteins. J Biol Chem 275, 33869–33875.[CrossRef]
    [Google Scholar]
  43. Nelson, K. E., Fouts, D. E., Mongodin, E. F., Ravel, J., DeBoy, R. T., Kolonay, J. F., Rasko, D. A., Angiuoli, S. V., Gill, S. R. & other authors ( 2004; ). Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res 32, 2386–2395.[CrossRef]
    [Google Scholar]
  44. Pancholi, V. & Chhatwal, G. S. ( 2003; ). Housekeeping enzymes as virulence factors for pathogens. Int J Med Microbiol 293, 391–401.[CrossRef]
    [Google Scholar]
  45. Pandiripally, V. K., Westbrook, D. G., Sunki, G. R. & Bhunia, A. K. ( 1999; ). Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2. J Med Microbiol 48, 117–124.[CrossRef]
    [Google Scholar]
  46. Pazos, M. J., Alfonso, A., Vieytes, M. R., Yasumoto, T., Vieites, J. M. & Botana, L. M. ( 2004; ). Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions. Anal Biochem 335, 112–118.[CrossRef]
    [Google Scholar]
  47. Pelech, S. ( 2004; ). Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions. Curr Pharm Biotechnol 5, 69–77.[CrossRef]
    [Google Scholar]
  48. Pentecost, M., Otto, G., Theriot, J. A. & Amieva, M. R. ( 2006; ). Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion. PLoS Pathog 2, e3.[CrossRef]
    [Google Scholar]
  49. Pilgrim, S., Kolb-Maurer, A., Gentschev, I., Goebel, W. & Kuhn, M. ( 2003; ). Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility. Infect Immun 71, 3473–3484.[CrossRef]
    [Google Scholar]
  50. Sabet, C., Toledo-Arana, A., Personnic, N., Lecuit, M., Dubrac, S., Poupel, O., Gouin, E., Nahori, M. A., Cossart, P. & Bierne, H. ( 2008; ). The Listeria monocytogenes virulence factor InlJ is specifically expressed in vivo and behaves as an adhesin. Infect Immun 76, 1368–1378.[CrossRef]
    [Google Scholar]
  51. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  52. Schaumburg, J., Diekmann, O., Hagendorff, P., Bergmann, S., Rohde, M., Hammerschmidt, S., Jansch, L., Wehland, J. & Karst, U. ( 2004; ). The cell wall subproteome of Listeria monocytogenes. Proteomics 4, 2991–3006.[CrossRef]
    [Google Scholar]
  53. Schubert, W. D., Urbanke, C., Ziehm, T., Beier, V., Machner, M. P., Domann, E., Wehland, J., Chakraborty, T. & Heinz, D. W. ( 2002; ). Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin. Cell 111, 825–836.[CrossRef]
    [Google Scholar]
  54. Scott, J. R. & Barnett, T. C. ( 2006; ). Surface proteins of gram-positive bacteria and how they get there. Annu Rev Microbiol 60, 397–423.[CrossRef]
    [Google Scholar]
  55. Seifert, K. N., McArthur, W. P., Bleiweis, A. S. & Brady, L. J. ( 2003; ). Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization, enzymatic activity, and protein-protein interactions. Can J Microbiol 49, 350–356.[CrossRef]
    [Google Scholar]
  56. Torian, B. E., Reed, S. L., Flores, B. M., Creely, C. M., Coward, J. E., Vial, K. & Stamm, W. E. ( 1990; ). The 96-kilodalton antigen as an integral membrane protein in pathogenic Entamoeba histolytica: potential differences in pathogenic and nonpathogenic isolates. Infect Immun 58, 753–760.
    [Google Scholar]
  57. Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J. & Kreft, J. ( 2001; ). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584–640.[CrossRef]
    [Google Scholar]
  58. Wampler, J. L., Kim, K. P., Jaradat, Z. & Bhunia, A. K. ( 2004; ). Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells. Infect Immun 72, 931–936.[CrossRef]
    [Google Scholar]
  59. Waseem, A., Yaqoob, M. & Nabi, A. ( 2006; ). Flow-injection determination of thyroxine using immobilized enzyme with tris (2,2′-bipyridyl)ruthenium(III) chemiluminescence detection. Anal Sci 22, 1095–1098.[CrossRef]
    [Google Scholar]
  60. Yang, W., Li, E., Kairong, T. & Stanley, S. L., Jr ( 1994; ). Entamoeba histolytica has an alcohol dehydrogenase homologous to the multifunctional adhE gene product of Escherichia coli. Mol Biochem Parasitol 64, 253–260.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.036509-0
Loading
/content/journal/micro/10.1099/mic.0.036509-0
Loading

Data & Media loading...

vol. , part 9, pp. 2782 - 2795

(including Figs S1-S3, and supplementary methods) [ PDF] (110 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