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
We have cloned and expressed in Escherichia coli a gene encoding a 15,000-apparent-molecular-weight peptidoglycan-associated outer membrane lipoprotein (PAL) of Haemophilus influenzae. The nucleotide sequence of this gene encodes an open reading frame of 153 codons with a predicted mature protein of 134 amino acids. The amino acid composition and sequence of the predicted mature protein agree with the chemically determined composition and partial amino acid sequence of PAL purified from H. influenzae outer membranes. We have also identified a second gene from H. influenzae that encodes a second 15,000-apparent-molecular-weight protein which is recognized by antiserum against PAL. This protein has been shown to be a lipoprotein. The nucleotide sequence of this gene encodes an open reading frame of 154 codons with a predicted mature protein of 136 amino acids and has limited sequence homology with that of the gene encoding PAL. Southern hybridization analysis indicates that both genes exist as single copies in H. influenzae chromosomal DNA. Both genes encode polypeptides which have amino-terminal sequences similar to those of reported membrane signal peptides and are associated primarily with the outer membrane when expressed in E. coli.
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