Analysis of pilus adhesins from Haemophilus influenzae biotype IV strains

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates f rom the human genital tract or from infected newborn infants forms a crypti c genospecies characterized by, among other features, the presence of perit richous pili. The objective of this study was to determine the similarity o f these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of h ifA and hifE and their gene products in NTHI biotype IV strains was assesse d, the binding of H. influenzae biotype IV strains to human epithelial cell s was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, w hich encodes the major structural subunit of hemagglutinating pili, and hif E, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected i n any of these strains by Western blot analysis using antisera that recogni ze HifA and HifE of respiratory strains. Transmission electron microscopy s howed no surface pili on the two biotype IV H. influenzae isolates examined ; strain 4162 containing an insertional mutation in hifA also showed no sur face pili, whereas strain 1595 containing an insertional mutation in hifB s howed pilus-like structures that were shorter and thicker than hemagglutina ting pili of the respiratory strains AAr176 and M43. In enzyme-linked immun osorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus rec eptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2 , or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did no t correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16H BE14o- and ME180 cells, hifA and hifB mutants of the biotype IV strains sho wed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-contain ing hemagglutinating pili, epithelial cell adherence exhibited by these str ains is not mediated by expression of hemagglutinating pili.