Cloning of chromosomal DNA from Haemophilus influenzae. Its use for studying the expression of type b capsule and virulence

J Clin Invest. 1984 Feb;73(2):298-306. doi: 10.1172/JCI111214.

Abstract

Haemophilus influenzae may make any one of six chemically distinct capsular polysaccharides, but only strains of capsular serotype b commonly cause systemic infection (e.g., meningitis) in humans. Molecular cloning of DNA was used to investigate the expression of type b capsule and its association with H. influenzae virulence. A virulent H. influenzae type b strain was used to construct a lambda library of chromosomal DNA in Charon 4. Two independently isolated recombinant phage were isolated from the library and were found to possess DNA necessary for expression of type b capsule. Using a well-characterized rat model of H. influenzae systemic infection, we showed that type b transformants elicited by the cloned DNA were pathogenic, causing bacteremia and meningitis, whereas the untransformed capsule-deficient H. influenzae organisms were not. A 4.4-kb EcoRI fragment, common to both DNA clones, was used to characterize clinical isolates representing all six encapsulated serotypes as well as several capsule-deficient H. influenzae by Southern hybridization analysis. The probe hybridized to an identical sized (4.4 kb) fragment of EcoRI-digested chromosomal DNA from eight independently isolated type b strains. Single bands of homology to the probe were also found in EcoRI fragments of chromosomal DNA obtained from 33 encapsulated, nontype b H. influenzae. However, the size of these EcoRI fragments proved to be characteristic for each of the different capsular serotypes. These studies provide a basis for pursuing the molecular analysis of the epidemiology and virulence of pathogenic H. influenzae.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cloning, Molecular*
  • DNA, Bacterial / genetics*
  • DNA, Recombinant
  • Haemophilus influenzae / classification
  • Haemophilus influenzae / genetics
  • Haemophilus influenzae / pathogenicity*
  • Mutation
  • Nucleic Acid Hybridization
  • Polysaccharides, Bacterial / genetics*
  • Polysaccharides, Bacterial / physiology
  • Rats
  • Rats, Inbred Strains

Substances

  • DNA, Bacterial
  • DNA, Recombinant
  • Polysaccharides, Bacterial