IDENTIFICATION OF A 2ND FAMILY OF HIGH-MOLECULAR-WEIGHT ADHESION PROTEINS EXPRESSED BY NONTYPABLE HAEMOPHILUS-INFLUENZAE

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of non-typab le Haemophilus influenzae (NTHI), mediate attachment to human epitheli al cells. These proteins are members of a family of highly immunogenic proteins common to 70-75% of NTHI strains. NTHI strains that lack HMW 1/HMW2-like proteins remain capable of efficient attachment to culture d human epithelial cells, suggesting the existence of additional adhes ion molecules. We reasoned that characterization of high-molecular-wei ght immunogenic proteins from an HMW1/HMW2-deficient strain might iden tify additional adhesion proteins. A genomic library was prepared in l ambda EMBL3 with chromosomal DNA from non-typable Haemophilus strain 1 1, a strain that lacks HMW1/HMW2-like proteins. The library was screen ed immunologically with convalescent serum from a child naturally infe cted with strain 11, and phage clones expressing high-molecular-weight recombinant proteins were identified by Western blot analysis. One cl one was identified that expressed a protein with an apparent molecular mass greater than 200 kDa. Transformation of non-adherent Escherichia coli strain DH5 alpha with plasmids containing the genetic locus enco ding this protein gave rise to E. coli transformants that adhered avid ly to Chang conjunctival cells. Subcloning and mutagenesis studies loc alized the DNA conferring the adherence phenotype to a 4.8 kbp fragmen t, and nucleotide sequence analysis further localized the gene encodin g the adhesion protein to a 3.3 kbp open reading frame predicted to en code a protein of 114 kDa. The gene was designated hia for Haemophilus influenzae adhesin. Southern analysis revealed an hia homologue in 13 of 15 HMW1/HMW2-deficient non-typable H. influenzae strains. In contr ast, the hia gene was not present in any of 23 non-typable H. influenz ae strains which expressed HMW1/HMW2-like proteins. Identification of this second family of high-molecular-weight adhesion proteins suggests the possibility of developing vaccines based upon a combination of HM W1/HMW2-like proteins and Hia-like proteins which would be protective against disease caused by most or all non-typable H. influenzae.