Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1990 Apr;58(4):903–908. doi: 10.1128/iai.58.4.903-908.1990

Cloning and expression in Escherichia coli of LKP pilus genes from a nontypeable Haemophilus influenzae strain.

S Kar 1, S C To 1, C C Brinton Jr 1
PMCID: PMC258558  PMID: 1969397

Abstract

Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C. C. To, S. C. M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C. M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C. C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity.

Full text

PDF
903

Images in this article

906Image
on p.906
906Image
on p.906
907Image
on p.907
907Image
on p.907

Selected References

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

  1. Anderson P. W., Pichichero M. E., Connor E. M. Enhanced nasopharyngeal colonization of rats by piliated Haemophilus influenzae type b. Infect Immun. 1985 May;48(2):565–568. doi: 10.1128/iai.48.2.565-568.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BRINTON C. C., Jr Non-flagellar appendages of bacteria. Nature. 1959 Mar 21;183(4664):782–786. doi: 10.1038/183782a0. [DOI] [PubMed] [Google Scholar]
  3. Brinton C. C., Jr The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans N Y Acad Sci. 1965 Jun;27(8):1003–1054. doi: 10.1111/j.2164-0947.1965.tb02342.x. [DOI] [PubMed] [Google Scholar]
  4. Hanson M. S., Brinton C. C., Jr Identification and characterization of E. coli type-1 pilus tip adhesion protein. Nature. 1988 Mar 17;332(6161):265–268. doi: 10.1038/332265a0. [DOI] [PubMed] [Google Scholar]
  5. Hanson M. S., Hempel J., Brinton C. C., Jr Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein. J Bacteriol. 1988 Aug;170(8):3350–3358. doi: 10.1128/jb.170.8.3350-3358.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Klemm P., Christiansen G. Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae. Mol Gen Genet. 1987 Jul;208(3):439–445. doi: 10.1007/BF00328136. [DOI] [PubMed] [Google Scholar]
  7. Klemm P., Jørgensen B. J., van Die I., de Ree H., Bergmans H. The fim genes responsible for synthesis of type 1 fimbriae in Escherichia coli, cloning and genetic organization. Mol Gen Genet. 1985;199(3):410–414. doi: 10.1007/BF00330751. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Levine M. M., Ristaino P., Marley G., Smyth C., Knutton S., Boedeker E., Black R., Young C., Clements M. L., Cheney C. Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans. Infect Immun. 1984 May;44(2):409–420. doi: 10.1128/iai.44.2.409-420.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lindberg F., Lund B., Johansson L., Normark S. Localization of the receptor-binding protein adhesin at the tip of the bacterial pilus. Nature. 1987 Jul 2;328(6125):84–87. doi: 10.1038/328084a0. [DOI] [PubMed] [Google Scholar]
  11. Loeb M. R., Connor E., Penney D. A comparison of the adherence of fimbriated and nonfimbriated Haemophilus influenzae type b to human adenoids in organ culture. Infect Immun. 1988 Feb;56(2):484–489. doi: 10.1128/iai.56.2.484-489.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Loos B. G., Bernstein J. M., Dryja D. M., Murphy T. F., Dickinson D. P. Determination of the epidemiology and transmission of nontypable Haemophilus influenzae in children with otitis media by comparison of total genomic DNA restriction fingerprints. Infect Immun. 1989 Sep;57(9):2751–2757. doi: 10.1128/iai.57.9.2751-2757.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maurer L., Orndorff P. E. Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili. J Bacteriol. 1987 Feb;169(2):640–645. doi: 10.1128/jb.169.2.640-645.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Minion F. C., Abraham S. N., Beachey E. H., Goguen J. D. The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit. J Bacteriol. 1986 Mar;165(3):1033–1036. doi: 10.1128/jb.165.3.1033-1036.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  16. Pichichero M. E. Adherence of Haemophilus influenzae to human buccal and pharyngeal epithelial cells: relationship to pilation. J Med Microbiol. 1984 Aug;18(1):107–116. doi: 10.1099/00222615-18-1-107. [DOI] [PubMed] [Google Scholar]
  17. Pichichero M. E., Loeb M., Anderson, Smith D. H. Do pili play a role in pathogenicity of Haemophilus influenzae type B? Lancet. 1982 Oct 30;2(8305):960–962. doi: 10.1016/s0140-6736(82)90161-1. [DOI] [PubMed] [Google Scholar]
  18. Purcell B. K., Clegg S. Construction and expression of recombinant plasmids encoding type 1 fimbriae of a urinary Klebsiella pneumoniae isolate. Infect Immun. 1983 Mar;39(3):1122–1127. doi: 10.1128/iai.39.3.1122-1127.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sable N. S., Connor E. M., Hall C. B., Loeb M. R. Variable adherence of fimbriated Haemophilus influenzae type b to human cells. Infect Immun. 1985 Apr;48(1):119–123. doi: 10.1128/iai.48.1.119-123.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. To S. C. F41 antigen among porcine enterotoxigenic Escherichia coli strains lacking K88, K99, and 987P pili. Infect Immun. 1984 Feb;43(2):549–554. doi: 10.1128/iai.43.2.549-554.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wong K., Roberts M. C., Smith A. L. The activity of Sch 29482 against type b Haemophilus influenzae lacking or possessing detectable beta-lactamase activity. J Antimicrob Chemother. 1982 Feb;9 (Suppl 100):163–170. doi: 10.1093/jac/9.suppl_c.163. [DOI] [PubMed] [Google Scholar]
  22. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  23. van Alphen L., Poole J., Geelen L., Zanen H. C. The erythrocyte and epithelial cell receptors for Haemophilus influenzae are expressed independently. Infect Immun. 1987 Oct;55(10):2355–2358. doi: 10.1128/iai.55.10.2355-2358.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES