Virulence determinants of pathogenic nontypeable Haemophilus influenzae
Fernaays, Matthew Michael
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Nontypeable Haemophilus influenzae is an important respiratory pathogen, causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). This bacterium possesses a high degree of genetic diversity, even among genes associated with virulence. Strains of H. influenzae are also associated with diverse clinical outcomes, especially with respect to COPD. Some strains are associated with exacerbation, or worsening of disease, while others colonize the host asymptomatically. To test the hypothesis that genetic differences among strains account for differences in pathogenic potential, competitive hybridization was performed using 6 strains associated with exacerbation of COPD and 5 strains associated with asymptomatic colonization with a microarray constructed from genomic fragments of an exacerbation strain. Seven sequences that were absent in all 5 colonization strains and present in at least 2 exacerbation strains were identified. To assess the distribution of the 7 sequences among well-characterized strains of H. influenzae, 132 strains were screened for the presence of these sequences. The presence or absence of any single sequence was not significantly associated with exacerbations of COPD. However, logistic regression and subgroup analysis identified combinations of the presence and absence of genes that are associated with exacerbations. One of the seven sequences identified was a previously unreported IgA protease gene, designated igaB. IgA protease is a well-described protein and potential virulence factor in this organism, as well as other respiratory pathogens. igaB displays homology to the neisserial IgA protease genes and is distinct from the previously described H. influenzae IgA protease gene, iga. Reverse transcriptase-PCR, and IgA protease assays indicated that the gene is transcribed, expressed, and enzymatically active in H. influenzae. Mutation analysis of iga and igaB indicated that igaB is the primary mediator of IgA protease activity in this strain. Examination of 297 strains of H. influenzae indicated that igaB is present in one-third of strains and is associated with strains causing clinical diseases compared to nasopharyngeal colonizers. It is also associated with increased levels of IgA protease activity. These studies indicated that patterns of genes are associated with the ability of strains of H. influenzae to cause exacerbations of COPD, supporting the concept that differences in pathogenic potential are based in part on genomic differences among infecting strains, not merely host factors. In addition, they support the hypothesis that the newly discovered igaB gene is a potential virulence factor in nontypeable H. influenzae.