Cloning of chromosomal DNA from Haemophilus influenzae. Its use for studying the expression of type b capsule and virulence.
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.
D000818:Animals; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant; D006193:Haemophilus influenzae / Q000145:classification / Q000235:genetics / Q000472:pathogenicity*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D011135:Polysaccharides, Bacterial / Q000235:genetics* / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D011135:Polysaccharides, Bacterial