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Infection and Immunity, February 2006, p. 1161-1170, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1161-1170.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Conservation and Diversity of HMW1 and HMW2 Adhesin Binding Domains among Invasive Nontypeable Haemophilus influenzae Isolates

Department of Infectious, Parasitic and Immune-Mediated Diseases,¹ Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy²

Received 9 May 2005/ Returned for modification 5 October 2005/ Accepted 2 November 2005

The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the hmw1A-like and hmw2A-like structural genes in nine invasive NTHi isolates, discovering two new hmwA alleles, whose sequences are herein reported. Then, the hmw1A-like and hmw2A-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.

Editor: V. J. DiRita

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