1887

Abstract

is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. analyses with CBU1224a showed no significant similarity to sequences outside strains of . Additional analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni or Zn, and roughly halved in the presence of Co or Fe. The gene was maximally expressed at 3–7 days post-infection in infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.046649-0
2011-04-01
2021-02-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/4/966.html?itemId=/content/journal/micro/10.1099/mic.0.046649-0&mimeType=html&fmt=ahah

References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A. Struhl K. editors 1995 Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  2. Babu M. M., Priya M. L., Selvan A. T., Madera M., Gough J., Aravind L., Sankaran K. 2006; A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins. J Bacteriol 188:2761–2773
    [Google Scholar]
  3. Beare P. A., Unsworth N., Andoh M., Voth D. E., Omsland A., Gilk S. D., Williams K. P., Sobral B. W., Kupko J. J. III other authors 2009; Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella. Infect Immun 77:642–656
    [Google Scholar]
  4. Chung C. T., Niemela S. L., Miller R. H. 1989; One-step preparation of competent Escherichia coli : transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86:2172–2175
    [Google Scholar]
  5. Cockrell D. C., Beare P. A., Fischer E. R., Howe D., Heinzen R. A. 2008; A method for purifying obligate intracellular Coxiella burnetii that employs digitonin lysis of host cells. J Microbiol Methods 72:321–325
    [Google Scholar]
  6. Coleman S. A., Fischer E. R., Howe D., Mead D. J., Heinzen R. A. 2004; Temporal analysis of Coxiella burnetii morphological differentiation. J Bacteriol 186:7344–7352
    [Google Scholar]
  7. Flores-Ramírez G., Toman R., Sekeyova Z., Skultety L. 2009; In silico prediction and identification of outer membrane proteins and lipoproteins from Coxiella burnetii by the mass spectrometry techniques. Clin Microbiol Infect 15:Suppl. 2196–197
    [Google Scholar]
  8. Fortney K. R., Young R. S., Bauer M. E., Katz B. P., Hood A. F., Munson R. S. Jr, Spinola S. M. 2000; Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection. Infect Immun 68:6441–6448
    [Google Scholar]
  9. Ge Y., Rikihisa Y. 2007; Surface-exposed proteins of Ehrlichia chaffeensis . Infect Immun 75:3833–3841
    [Google Scholar]
  10. Geukens N., De Buck E., Meyen E., Maes L., Vranckx L., Van Mellaert L., , Anné J., Lammertyn E. 2006; The type II signal peptidase of Legionella pneumophila . Res Microbiol 157:836–841
    [Google Scholar]
  11. Hantke K., Braun V. 1973; Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem 34:284–296
    [Google Scholar]
  12. Hayashi S., Wu H. C. 1990; Lipoproteins in bacteria. J Bioenerg Biomembr 22:451–471
    [Google Scholar]
  13. Hicks L. D., Raghavan R., Battisti J. M., Minnick M. F. 2010; A DNA-binding peroxiredoxin of Coxiella burnetii is involved in countering oxidative stress during exponential-phase growth. J Bacteriol 192:2077–2084
    [Google Scholar]
  14. Hoover T. A., Culp D. W., Vodkin M. H., Williams J. C., Thompson H. A. 2002; Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain. Infect Immun 70:6726–6733
    [Google Scholar]
  15. Hutchings M. I., Palmer T., Harrington D. J., Sutcliffe I. C. 2009; Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em. Trends Microbiol 17:13–21
    [Google Scholar]
  16. Inouye S., Wang S., Sekizawa J., Halegoua S., Inouye M. 1977; Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc Natl Acad Sci U S A 74:1004–1008
    [Google Scholar]
  17. Kiho T., Nakayama M., Yasuda K., Miyakoshi S., Inukai M., Kogen H. 2004; Structure–activity relationships of globomycin analogues as antibiotics. Bioorg Med Chem 12:337–361
    [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  19. Maurin M., Raoult D. 1999; Q fever. Clin Microbiol Rev 12:518–553
    [Google Scholar]
  20. McCaul T. F., Williams J. C. 1981; Developmental cycle of Coxiella burnetii : structure and morphogenesis of vegetative and sporogenic differentiations. J Bacteriol 147:1063–1076
    [Google Scholar]
  21. Moos A., Hackstadt T. 1987; Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model. Infect Immun 55:1144–1150
    [Google Scholar]
  22. Parrow N. L., Abbott J., Lockwood A. R., Battisti J. M., Minnick M. F. 2009; Function, regulation, and transcriptional organization of the hemin utilization locus of Bartonella quintana . Infect Immun 77:307–316
    [Google Scholar]
  23. Raghavan R., Hicks L. D., Minnick M. F. 2008; Toxic introns and parasitic intein in Coxiella burnetii : legacies of a promiscuous past. J Bacteriol 190:5934–5943
    [Google Scholar]
  24. Rahman M. S., Ceraul S. M., Dreher-Lesnick S. M., Beier M. S., Azad A. F. 2007; The lspA gene, encoding the type II signal peptidase of Rickettsia typhi : transcriptional and functional analysis. J Bacteriol 189:336–341
    [Google Scholar]
  25. Ratledge C., Dover L. G. 2000; Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54:881–941
    [Google Scholar]
  26. Regis E. 1999 The biology of doom: The history of America’s secret germ warfare project New York: Henry Holt & Co;
    [Google Scholar]
  27. Rosenzweig A. C. 2002; Metallochaperones: bind and deliver. Chem Biol 9:673–677
    [Google Scholar]
  28. Samoilis G., Psaroulaki A., Vougas K., Tselentis Y., Tsiotis G. 2007; Analysis of whole cell lysate from the intercellular bacterium Coxiella burnetii using two gel-based protein separation techniques. J Proteome Res 6:3032–3041
    [Google Scholar]
  29. Schwan T. G., Piesman J., Golde W. T., Dolan M. C., Rosa P. A. 1995; Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci U S A 92:2909–2913
    [Google Scholar]
  30. Seshadri R., Samuel J. 2005; Genome analysis of Coxiella burnetii species: insights into pathogenesis and evolution and implications for biodefense. Ann N Y Acad Sci 1063:442–450
    [Google Scholar]
  31. Seshadri R., Paulsen I. T., Eisen J. A., Read T. D., Nelson K. E., Nelson W. C., Ward N. L., Tettelin H., Davidsen T. M. other authors 2003; Complete genome sequence of the Q-fever pathogen Coxiella burnetii . Proc Natl Acad Sci U S A 100:5455–5460
    [Google Scholar]
  32. Sha J., Fadl A. A., Klimpel G. R., Niesel D. W., Popov V. L., Chopra A. K. 2004; The two murein lipoproteins of Salmonella enterica serovar Typhimurium contribute to the virulence of the organism. Infect Immun 72:3987–4003
    [Google Scholar]
  33. Shannon J. G., Howe D., Heinzen R. A. 2005; Virulent Coxiella burnetii does not activate human dendritic cells: role of lipopolysaccharide as a shielding molecule. Proc Natl Acad Sci U S A 102:8722–8727
    [Google Scholar]
  34. Slupska M. M., Chiang J. H., Luther W. M., Stewart J. L., Amii L., Conrad A., Miller J. H. 2000; Genes involved in the determination of the rate of inversions at short inverted repeats. Genes Cells 5:425–437
    [Google Scholar]
  35. Tokuda H. 2009; Biogenesis of outer membranes in Gram-negative bacteria. Biosci Biotechnol Biochem 73:465–473
    [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354
    [Google Scholar]
  37. Vodkin M. H., Williams J. C. 1986; Overlapping deletion in two spontaneous phase variants of Coxiella burnetii . J Gen Microbiol 132:2587–2594
    [Google Scholar]
  38. Voth D. E., Heinzen R. A. 2007; Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii . Cell Microbiol 9:829–840
    [Google Scholar]
  39. Yamaguchi K., Yu F., Inouye M. 1988; A single amino acid determinant of the membrane localization of lipoproteins in E. coli . Cell 53:423–432
    [Google Scholar]
  40. Zamboni D. S., McGrath S., Rabinovitch M., Roy C. R. 2003; Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system. Mol Microbiol 49:965–976
    [Google Scholar]
  41. Zhang G., To H., Russell K. E., Hendrix L. R., Yamaguchi T., Fukushi H., Hirai K., Samuel J. E. 2005; Identification and characterization of an immunodominant 28-kilodalton Coxiella burnetii outer membrane protein specific to isolates associated with acute disease. Infect Immun 73:1561–1567
    [Google Scholar]
  42. Zhao H., Waite J. H. 2006; Proteins in load-bearing junctions: the histidine-rich metal-binding protein of mussel byssus. Biochemistry 45:14223–14231
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.046649-0
Loading
/content/journal/micro/10.1099/mic.0.046649-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

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