COMPARATIVE-ANALYSIS OF HAEMOPHILUS-INFLUENZAE HIFA (PILIN) GENES

Adherence of Haemophilus influenzae to epithelial cells plays a centra l role in colonization and is the first step in infection with this or ganism, pill, which are large polymorphic surface proteins, have been shown to mediate the binding of H. influenzae to cells of the human re spiratory tract. Earlier experiments have demonstrated that the major epitopes of H. influenzae pill are highly conformational and immunolog ically heterogenous; their subunit pilins are, however, immunologicall y homogenous. To define the extent of structural variation in pilins, which polymerize to form pill, the pilin genes (hifA) of 26 type a to f and 16 nontypeable strains of H. influenzae were amplified by PCR an d subjected to restriction fragment length polymorphism (RFLP) analysi s with AluI and RsaI. Six different RFLP patterns,were identified, Fou r further RFLP patterns were identified from published hifA sequences from five nontypeable H. influenzae strains. Two patterns contained on ly nontypeable isolates; one of these contained H. influenzae biotype aegyptius strains F3031 and F3037. Another pattern contained predomina ntly H. influenzae type f strains. All other patterns were displayed b y a variety of capsular and noncapsular types. Sequence analysis of se lected hifA genes confirmed the 10 RFLP patterns and showed strong ide ntity among representatives displaying the same RFLP patterns, In addi tion, the immunologic reactivity of pill with antipilus antisera corre lated with the groupings of strains based on hifA RFLP patterns. Those strains that show greater reactivity with antiserum directed against H. influenzae type b strain M43 pill tend to fall into one RFLP patter n (pattern 3); while those strains that show equal or greater reactivi ty with antiserum directed against H. influenzae type b strain Eagan p ill tend to fall in a different RFLP pattern (pattern 1). Sequence ana lysis of representative HifA pilins from typeable and nontypeable H. i nfluenzae identified several highly conserved regions that play a role in bacterial pilus assembly and other regions with considerable amino acid heterogeneity, These regions of HifA amino acid sequence heterog eneity may explain the immunologic diversity seen in intact pili.