Haemophilus ducreyi Secretes a Filamentous Hemagglutinin-Like Protein

Clustal-W alignment of selected regions of LspA1 and LspA2 with homologous proteins. The N-terminal 380 aa of LspA1 and LspA2 were aligned with the N-terminal region of FhaB (A). Internal regions of LspA1 and LspA2 were aligned with the C-terminal one-third of the H. somnus P76 protein (B). The shaded box highlights the NPNG(I/M) motif. Asterisks indicate identical amino acids; periods indicate conserved amino acid substitutions.

Schematic representation of the predicted protein products of H. ducreyi 35000 lspA1 andlspA2. Regions of nearly complete identity between LspA1 and LspA2 are displayed as boxes with identical shading. The locations of the amino acid sequences of LspA1 and LspA2 used to produce MAbs 40A4, 11B7, and 1H9 are indicated. The location of the region that has 70% identity with the H. somnus P76 protein is also indicated (hatched box), as is the location of the three contiguous 319-aa repeats present in LspA2.

Agarose gel electrophoresis of H. ducreyi multiplex RT-PCR products. The predicted sizes of theH. ducreyi 35000 RT-PCR products were as follows:pal, 354 bp; lspA1, 264 bp; and lspA2, 320 bp. The templates included in each of the reaction mixtures were as follows: lane 1, no template (negative control); lane 2, 100 ng ofH. ducreyi 35000 genomic DNA (positive control); lanes 3 and 7, 2 μg of RNA isolated from H. ducreyi grown on chocolate agar plates; lanes 4 and 8, 2 μg of RNA isolated fromH. ducreyi grown for 8 h in liquid broth; lanes 5 and 9, 2 μg of RNA isolated from H. ducreyi grown for 25 h in liquid broth; lanes 6 and 10, 3 μg of RNA isolated fromH. ducreyi-infected rabbit lesions. The reaction mixtures loaded in lanes 7 to 10 were not subjected to the reverse transcription step of the RT-PCR protocol and served as controls to detect DNA contamination of the RNA templates. Lanes M contain DNA size markers.

Western blot analysis of CCS from 12 H. ducreyi strains. CCS from each strain was probed with polyclonalH. ducreyi 35000-infected rabbit serum (A), MAb 11B7 (B), MAb 40A4 (C), and MAb 1H9 (D). Lanes: 1, uninoculated medium control; 2, 35000; 3, R018; 4, 181; 5, CA173; 6, WPB506; 7, BG411; 8, 041; 9, CIP542; 10, A77; 11, 6V; 12, E1673; 13, 78226. Rabbit antibodies were detected with a horseradish peroxidase-conjugated secondary antibody; MAbs were detected with radioiodinated secondary antibodies. The immunoreactive doublets apparent in panel A are the result of the tendency of LspA1 to give rise to multiple forms.