Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Oct;177(20):5790–5798. doi: 10.1128/jb.177.20.5790-5798.1995

Molecular analysis of a metalloprotease from Proteus mirabilis.

C Wassif 1, D Cheek 1, R Belas 1
PMCID: PMC177400  PMID: 7592325

Abstract

Proteus mirabilis is known for its ability to differentiate from swimmer to swarmer cells, a process crucial for the pathogenesis of these bacteria during urinary tract infections. Among the many virulence factors produced during swarmer cell differentiation is an extracellular metalloprotease. A cosmid containing a large fragment of P. mirabilis chromosomal DNA was obtained by measuring protease expression in recombinant Escherichia coli. The recombinant and native enzymes were purified to over 95% homogeneity from culture supernatants by use of phenyl-Sepharose affinity chromatography and found to be identical. The activity of the 55-kDa enzyme was stimulated by divalent cations (Ca2+ > Mg2+) and inhibited by a chelator of these cations. The enzyme possesses substrate specificity for both serum and secretory forms of immunoglobulin A1 (IgA1) and IgA2 as well as IgG and, unlike classic IgA proteases, digested to completion both human and mouse IgA. Following subcloning, a 5-kb DNA fragment encoding recombinant protease activity was identified by insertional mutagenesis with Tn5. Four open reading frames were identified within this 5-kb region by limited nucleotide sequence analysis of DNA flanking the transposon. The nucleotide and deduced amino acid sequences of the metalloprotease structural gene (zapA) were obtained. Computerized homology studies revealed that the P. mirabilis metalloprotein is a member of the serralysin family of proteases and may be part of an operon comprising genes encoding an ATP-dependent ABC transporter in addition to the metalloprotease. The relevance of the metalloprotease to swarmer cell differentiation and pathogenicity is discussed.

Full Text

The Full Text of this article is available as a PDF (352.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allison C., Coleman N., Jones P. L., Hughes C. Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation. Infect Immun. 1992 Nov;60(11):4740–4746. doi: 10.1128/iai.60.11.4740-4746.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allison C., Emödy L., Coleman N., Hughes C. The role of swarm cell differentiation and multicellular migration in the uropathogenicity of Proteus mirabilis. J Infect Dis. 1994 May;169(5):1155–1158. doi: 10.1093/infdis/169.5.1155. [DOI] [PubMed] [Google Scholar]
  3. Allison C., Lai H. C., Gygi D., Hughes C. Cell differentiation of Proteus mirabilis is initiated by glutamine, a specific chemoattractant for swarming cells. Mol Microbiol. 1993 Apr;8(1):53–60. doi: 10.1111/j.1365-2958.1993.tb01202.x. [DOI] [PubMed] [Google Scholar]
  4. Allison C., Lai H. C., Hughes C. Co-ordinate expression of virulence genes during swarm-cell differentiation and population migration of Proteus mirabilis. Mol Microbiol. 1992 Jun;6(12):1583–1591. doi: 10.1111/j.1365-2958.1992.tb00883.x. [DOI] [PubMed] [Google Scholar]
  5. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  6. Bassler B. L., Wright M., Showalter R. E., Silverman M. R. Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol. 1993 Aug;9(4):773–786. doi: 10.1111/j.1365-2958.1993.tb01737.x. [DOI] [PubMed] [Google Scholar]
  7. Bassler B. L., Wright M., Silverman M. R. Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi. Mol Microbiol. 1994 May;12(3):403–412. doi: 10.1111/j.1365-2958.1994.tb01029.x. [DOI] [PubMed] [Google Scholar]
  8. Baumann U. Crystal structure of the 50 kDa metallo protease from Serratia marcescens. J Mol Biol. 1994 Sep 23;242(3):244–251. doi: 10.1006/jmbi.1994.1576. [DOI] [PubMed] [Google Scholar]
  9. Belas R., Erskine D., Flaherty D. Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior. J Bacteriol. 1991 Oct;173(19):6279–6288. doi: 10.1128/jb.173.19.6279-6288.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Belas R., Erskine D., Flaherty D. Transposon mutagenesis in Proteus mirabilis. J Bacteriol. 1991 Oct;173(19):6289–6293. doi: 10.1128/jb.173.19.6289-6293.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Belas R. Expression of multiple flagellin-encoding genes of Proteus mirabilis. J Bacteriol. 1994 Dec;176(23):7169–7181. doi: 10.1128/jb.176.23.7169-7181.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Belas R., Flaherty D. Sequence and genetic analysis of multiple flagellin-encoding genes from Proteus mirabilis. Gene. 1994 Oct 11;148(1):33–41. doi: 10.1016/0378-1119(94)90230-5. [DOI] [PubMed] [Google Scholar]
  13. Belas R., Goldman M., Ashliman K. Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation. J Bacteriol. 1995 Feb;177(3):823–828. doi: 10.1128/jb.177.3.823-828.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Bellalou J., Sakamoto H., Ladant D., Geoffroy C., Ullmann A. Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase. Infect Immun. 1990 Oct;58(10):3242–3247. doi: 10.1128/iai.58.10.3242-3247.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Burrows L. L., Lo R. Y. Molecular characterization of an RTX toxin determinant from Actinobacillus suis. Infect Immun. 1992 Jun;60(6):2166–2173. doi: 10.1128/iai.60.6.2166-2173.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chang Y. F., Young R., Struck D. K. Cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae. DNA. 1989 Nov;8(9):635–647. doi: 10.1089/dna.1.1989.8.635. [DOI] [PubMed] [Google Scholar]
  17. Coetzee J. N. Genetics of the Proteus group. Annu Rev Microbiol. 1972;26:23–54. doi: 10.1146/annurev.mi.26.100172.000323. [DOI] [PubMed] [Google Scholar]
  18. Cruz W. T., Young R., Chang Y. F., Struck D. K. Deletion analysis resolves cell-binding and lytic domains of the Pasteurella leukotoxin. Mol Microbiol. 1990 Nov;4(11):1933–1939. doi: 10.1111/j.1365-2958.1990.tb02042.x. [DOI] [PubMed] [Google Scholar]
  19. Delepelaire P., Wandersman C. Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the gram-negative bacterium Erwinia chrysanthemi. Mol Microbiol. 1991 Oct;5(10):2427–2434. doi: 10.1111/j.1365-2958.1991.tb02088.x. [DOI] [PubMed] [Google Scholar]
  20. Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide. J Biol Chem. 1989 May 25;264(15):9083–9089. [PubMed] [Google Scholar]
  21. Delepelaire P., Wandersman C. Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin. J Biol Chem. 1990 Oct 5;265(28):17118–17125. [PubMed] [Google Scholar]
  22. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Duong F., Lazdunski A., Cami B., Murgier M. Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways. Gene. 1992 Nov 2;121(1):47–54. doi: 10.1016/0378-1119(92)90160-q. [DOI] [PubMed] [Google Scholar]
  24. Duong F., Soscia C., Lazdunski A., Murgier M. The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system. Mol Microbiol. 1994 Mar;11(6):1117–1126. doi: 10.1111/j.1365-2958.1994.tb00388.x. [DOI] [PubMed] [Google Scholar]
  25. Döring G., Obernesser H. J., Botzenhart K. Extrazelluläre Toxine von Pseudomonas aeruginosa. II. Einwirkung zweier gereinigter Proteasen auf die menschlichen Immunoglobuline IgG, IgA und sekretorisches IgA. Zentralbl Bakteriol A. 1981 Mar;249(1):89–98. [PubMed] [Google Scholar]
  26. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  27. Ghigo J. M., Wandersman C. A carboxyl-terminal four-amino acid motif is required for secretion of the metalloprotease PrtG through the Erwinia chrysanthemi protease secretion pathway. J Biol Chem. 1994 Mar 25;269(12):8979–8985. [PubMed] [Google Scholar]
  28. Ghigo J. M., Wandersman C. 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. 1992 Dec;236(1):135–144. doi: 10.1007/BF00279652. [DOI] [PubMed] [Google Scholar]
  29. Gish W., States D. J. Identification of protein coding regions by database similarity search. Nat Genet. 1993 Mar;3(3):266–272. doi: 10.1038/ng0393-266. [DOI] [PubMed] [Google Scholar]
  30. Gygi D., Bailey M. J., Allison C., Hughes C. Requirement for FlhA in flagella assembly and swarm-cell differentiation by Proteus mirabilis. Mol Microbiol. 1995 Feb;15(4):761–769. doi: 10.1111/j.1365-2958.1995.tb02383.x. [DOI] [PubMed] [Google Scholar]
  31. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  32. Holland I. B., Kenny B., Blight M. Haemolysin secretion from E coli. Biochimie. 1990 Feb-Mar;72(2-3):131–141. doi: 10.1016/0300-9084(90)90138-7. [DOI] [PubMed] [Google Scholar]
  33. Johnson D. E., Russell R. G., Lockatell C. V., Zulty J. C., Warren J. W., Mobley H. L. Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection. Infect Immun. 1993 Jul;61(7):2748–2754. doi: 10.1128/iai.61.7.2748-2754.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Jones B. D., Mobley H. L. Proteus mirabilis urease: genetic organization, regulation, and expression of structural genes. J Bacteriol. 1988 Aug;170(8):3342–3349. doi: 10.1128/jb.170.8.3342-3349.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kornfeld S. J., Plaut A. G. Secretory immunity and the bacterial IgA proteases. Rev Infect Dis. 1981 May-Jun;3(3):521–534. doi: 10.1093/clinids/3.3.521. [DOI] [PubMed] [Google Scholar]
  36. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  37. Lally E. T., Golub E. E., Kieba I. R., Taichman N. S., Rosenbloom J., Rosenbloom J. C., Gibson C. W., Demuth D. R. Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins. J Biol Chem. 1989 Sep 15;264(26):15451–15456. [PubMed] [Google Scholar]
  38. Lally E. T., Kieba I. R., Demuth D. R., Rosenbloom J., Golub E. E., Taichman N. S., Gibson C. W. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem Biophys Res Commun. 1989 Feb 28;159(1):256–262. doi: 10.1016/0006-291x(89)92431-5. [DOI] [PubMed] [Google Scholar]
  39. Loomes L. M., Kerr M. A., Senior B. W. The cleavage of immunoglobulin G in vitro and in vivo by a proteinase secreted by the urinary tract pathogen Proteus mirabilis. J Med Microbiol. 1993 Sep;39(3):225–232. doi: 10.1099/00222615-39-3-225. [DOI] [PubMed] [Google Scholar]
  40. Loomes L. M., Senior B. W., Kerr M. A. A proteolytic enzyme secreted by Proteus mirabilis degrades immunoglobulins of the immunoglobulin A1 (IgA1), IgA2, and IgG isotypes. Infect Immun. 1990 Jun;58(6):1979–1985. doi: 10.1128/iai.58.6.1979-1985.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Loomes L. M., Senior B. W., Kerr M. A. Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients. Infect Immun. 1992 Jun;60(6):2267–2273. doi: 10.1128/iai.60.6.2267-2273.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Létoffé S., Delepelaire P., Wandersman C. 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. 1991 Apr;173(7):2160–2166. doi: 10.1128/jb.173.7.2160-2166.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Létoffé S., Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin. EMBO J. 1990 May;9(5):1375–1382. doi: 10.1002/j.1460-2075.1990.tb08252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Létoffé S., Ghigo J. M., Wandersman C. Identification of two components of the Serratia marcescens metalloprotease transporter: protease SM secretion in Escherichia coli is TolC dependent. J Bacteriol. 1993 Nov;175(22):7321–7328. doi: 10.1128/jb.175.22.7321-7328.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Milazzo F. H., Delisle G. J. Immunoglobulin A proteases in gram-negative bacteria isolated from human urinary tract infections. Infect Immun. 1984 Jan;43(1):11–13. doi: 10.1128/iai.43.1.11-13.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mobley H. L., Belas R. Swarming and pathogenicity of Proteus mirabilis in the urinary tract. Trends Microbiol. 1995 Jul;3(7):280–284. doi: 10.1016/s0966-842x(00)88945-3. [DOI] [PubMed] [Google Scholar]
  47. Mobley H. L., Warren J. W. Urease-positive bacteriuria and obstruction of long-term urinary catheters. J Clin Microbiol. 1987 Nov;25(11):2216–2217. doi: 10.1128/jcm.25.11.2216-2217.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Molla A., Kagimoto T., Maeda H. Cleavage of immunoglobulin G (IgG) and IgA around the hinge region by proteases from Serratia marcescens. Infect Immun. 1988 Apr;56(4):916–920. doi: 10.1128/iai.56.4.916-920.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nakahama K., Yoshimura K., Marumoto R., Kikuchi M., Lee I. S., Hase T., Matsubara H. Cloning and sequencing of Serratia protease gene. Nucleic Acids Res. 1986 Jul 25;14(14):5843–5855. doi: 10.1093/nar/14.14.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Plaut A. G. The IgA1 proteases of pathogenic bacteria. Annu Rev Microbiol. 1983;37:603–622. doi: 10.1146/annurev.mi.37.100183.003131. [DOI] [PubMed] [Google Scholar]
  51. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Senior B. W., Albrechtsen M., Kerr M. A. A survey of IgA protease production among clinical isolates of Proteeae. J Med Microbiol. 1988 Jan;25(1):27–31. doi: 10.1099/00222615-25-1-27. [DOI] [PubMed] [Google Scholar]
  53. Senior B. W., Albrechtsen M., Kerr M. A. Proteus mirabilis strains of diverse type have IgA protease activity. J Med Microbiol. 1987 Sep;24(2):175–180. doi: 10.1099/00222615-24-2-175. [DOI] [PubMed] [Google Scholar]
  54. Senior B. W., Loomes L. M., Kerr M. A. The production and activity in vivo of Proteus mirabilis IgA protease in infections of the urinary tract. J Med Microbiol. 1991 Oct;35(4):203–207. doi: 10.1099/00222615-35-4-203. [DOI] [PubMed] [Google Scholar]
  55. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  56. Showalter R. E., Martin M. O., Silverman M. R. Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi. J Bacteriol. 1990 Jun;172(6):2946–2954. doi: 10.1128/jb.172.6.2946-2954.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tan Y., Miller K. J. Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52. Appl Environ Microbiol. 1992 Apr;58(4):1402–1407. doi: 10.1128/aem.58.4.1402-1407.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Thompson S. A., Wang L. L., West A., Sparling P. F. Neisseria meningitidis produces iron-regulated proteins related to the RTX family of exoproteins. J Bacteriol. 1993 Feb;175(3):811–818. doi: 10.1128/jb.175.3.811-818.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tommassen J., Filloux A., Bally M., Murgier M., Lazdunski A. Protein secretion in Pseudomonas aeruginosa. FEMS Microbiol Rev. 1992 Sep;9(1):73–90. doi: 10.1016/0378-1097(92)90336-m. [DOI] [PubMed] [Google Scholar]
  60. Wandersman C. Secretion across the bacterial outer membrane. Trends Genet. 1992 Sep;8(9):317–322. doi: 10.1016/0168-9525(92)90264-5. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES