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

Abstract

As bstract. 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 ofchromosomal 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 A preliminary report ofthis work was presented at the Society

Presence of Copper- and Zinc-Containing Superoxide Dismutase in Commensal Haemophilus haemolyticus Isolates Can Be Used as a Marker To Discriminate Them from Nontypeable H. influenzae Isolates

“…N. meningitidis was cultured as described previously (27). Chromosomal DNA was prepared from the plate-grown organisms (17), and standard methods were used for restriction endonuclease digestion and Southern blotting, with washing to 80% stringency (24). PCR amplification of genomic DNA with the degenerate primers 3ЈUNIVSOD and 5ЈUNIVSOD, designed to amplify a 300-bp conserved region of prokaryotic sodC genes, was performed as described previously (12), except that an annealing temperature of 44°C was used.…”

Capsulation gene loss and 'rescue' mutations during the Cap+ to Cap- transition in Haemophilus influenzae type b

“…H . injluenzae was made competent for the uptake of linearized DNA by growth in sBHI broth followed by incubation in MIV medium (Herriott et al, 1970) as described previously (Moxon et al, 1984). …”

“…injluenzae chromosomal DNA. Total cellular DNA was prepared from 3ml broth cultures as previously described (Moxon et al, 1984). Genetic analysis of variants and transformants was accomplished by digestion of total cellular DNA with EcoRI, electrophoresis in agarose/Tris acetate gels and Southern blotting.…”

“…More than 95% of systemic infections caused by H. influenzae are due to organisms possessing the type b capsule (Turk, 1982), a polymer of ribose and ribitol5-phosphate (PRP), which is an important virulence determinant (Moxon et al, 1984).…”

Copper-zinc superoxide dismutase of Haemophilus influenzae and H. parainfluenzae

“…Total cellular DNA was prepared from 3-ml broth cultures of Haemophilus strains as previously described (32). Standard methods were used for restriction digestion, Southern blotting, preparation of plasmid DNA, colony hybridization, and 5' end labeling of oligonucleotides (30).…”