Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae.
A Haemophilus influenzae b (Hib) membrane protein with a molecular mass of 28 kDa bound polyclonal antisera raised against a highly purified Hib fimbrial subunit. We cloned the gene encoding this protein and found that the gene was expressed in Escherichia coli. DNA sequence analysis identified an 843-bp open reading frame which predicted a 26.78-kDa protein with an amino-terminal signal sequence and a mature protein with 70% similarity to the 28-kDa lipoprotein of E. coli (F. Yu, S. Inouye, and M. Inouye, J. Biol. Chem. 261:2284, 1986). Colony blot hybridization analysis with an intergenic probe of the cloned gene demonstrated that 29 of 32 H. influenzae strains hybridize with this gene. Insertion of a chloramphenicol acetyltransferase gene into the open reading frame inactivated expression of the 28-kDa protein in E. coli. Isogenic Hib strains were derived by marker exchange mutagenesis to generate mutants which no longer expressed the 28-kDa protein as recognized with Western immunoblot analysis. There was no difference in the rate of nasopharyngeal colonization of infant rats or monkeys by the isogenic mutants which lacked the 28-kDa protein compared with colonization by the wild-type strain. In contrast, the frequency of invasion and density of bacteremia in infant rats caused by the isogenic mutants were reduced relative to those caused by the wild-type Hib strain. We conclude that this 28-kDa outer membrane protein aids transepithelial invasion of type b strains but is not essential.
Chanyangam
M
M
Department of Pediatrics, School of Medicine, University of Washington, Seattle 98105.
Smith
A L
AL
Moseley
S L
SL
Kuehn
M
M
Jenny
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005260:Female; D006193:Haemophilus influenzae / Q000472:pathogenicity*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000633:toxicity*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011817:Rabbits; D014774:Virulence
D008565:Membrane Proteins