FINE TANGLED PILI EXPRESSED BY HAEMOPHILUS-DUCREYI ARE A NOVEL CLASS OF PILI

Haemophilus ducreyi synthesizes fine, tangled pill composed predominan tly of a protein whose apparent molecular weight is 24,000 (24K). A hy bridoma, 2D8, produced a monoclonal antibody (MAb) that bound to a 24K protein in H. ducreyi strains isolated from diverse geographic locati ons. A lambda gt11 H. ducreyi library was screened with MAb 2D8. A 3.5 -kb chromosomal insert from one reactive plaque was amplified and liga ted into the pCRII vector. The recombinant plasmid, designated pHD24, expressed a 24K protein in Escherichia coli INV alpha F' that bound MA b 2D8. The coding sequence of the 24K gene was localized by exonucleas e III digestion. The insert contained a 570-bp open reading frame, des ignated ftpA (fine, tangled pill). Translation of ftpA predicted a pol ypeptide with a molecular weight of 21.1K. The predicted N-terminal am ino acid sequence of the polypeptide encoded by ftpA was identical to the N-terminal amino acid sequence of purified pilin and lacked a clea vable signal sequence. Primer extension analysis of ftpA confirmed the lack of a leader peptide. The predicted amino acid sequence lacked ho mology to known pilin sequences but shared homology,vith the sequences of E. coli Dps and Treponema pallidum antigen TpF1 or 4D, proteins wh ich associate to form ordered rings. An isogenic pilin mutant, H. ducr eyi 35000ftpA::mTn3(Cm), was constructed by shuttle mutagenesis and di d not contain pill when examined by electron microscopy. We conclude t hat H. ducreyi synthesizes fine, tangled pill that are composed of a u nique major subunit, which may be exported by a signal sequence indepe ndent mechanism.