Analysis of Pilus Adhesins from Haemophilus influenzae Biotype IV Strains

Abstract

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA-and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells 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, which encodes the major structural subunit of hemagglutinating pili, and hifE, 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 in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent 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 receptor 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 not 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 16HBE14o ؊ and ME180 cells, hifA and hifB mutants of the biotype IV strains showed 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-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.Nontypeable Haemophilus influenzae (NTHI; i.e., strains lacking a polysaccharide capsule) strains colonize the human pharynx and nasopharynx and may cause respiratory infections such as otitis media, bronchitis, sinusitis, and pneumonia...

Identification of a Novel Trimeric Autotransporter Adhesin in the Cryptic Genospecies of Haemophilus

“…As shown in Fig. 1, strains 422, 1595, and 1673 were highly adherent to all of these cell lines, consistent with published results (9,38). The fourth clinical isolate, strain 420, was nonadherent with all of the cell lines.…”

“…As a first step toward understanding the tissue tropism of the Haemophilus cryptic genospecies, we extended previous studies by Clemans et al and Rosenau et al and examined the adherence of four strains to both genital (HeLa and HEC-1-B) and respiratory (Chang and Detroit 562) epithelial cell lines (9,38). As shown in Fig.…”

“…Initial work suggested that these fibers might be related to H. influenzae hemagglutinating pili, which are encoded by the hif gene cluster, which consists of five genes designated hifA, hifB, hifC, hifD, and hifE. In particular, PCR analysis has demonstrated that a subset of Haemophilus cryptic genospecies isolates contain hif-like genes (4,9,17). However, antisera against the H. influenzae HifA and HifE proteins are nonreactive with these isolates (9).…”

“…In particular, PCR analysis has demonstrated that a subset of Haemophilus cryptic genospecies isolates contain hif-like genes (4,9,17). However, antisera against the H. influenzae HifA and HifE proteins are nonreactive with these isolates (9). Furthermore, the short, thin surface fibers observed in the Haemophilus cryptic genospecies differ in appearance from H. influenzae hemagglutinating pili, which are longer and thicker (4).…”

Nanoscale Structural and Mechanical Properties of Nontypeable Haemophilus influenzae Biofilms

“…Fimbriae of different strains of H. influenzae bacteria have been visualized in great detail using electron microscopy (EM), particularly transmission EM (TEM) (10,20,33,41,52,69). In addition, their LOS composition has been extensively investigated by electrophoretic and mass spectrometric methods (11,15,38,39,44).…”