Figure 1 Identification of the ompP4 gene within H. ducreyi 35000HP. A, Map of the ompP4–containing locus. B, PCR amplification of the ompP4 locus from genomic DNA of ten clinical
isolates. Lanes 1–6, class I strains 35000HP, HD183, HD188, 82–029362, 6644, and 85–023233, respectively; lanes 7–10, class II strains CIP542 TCC, DMC64, 33921 and HMC112, respectively; Evofosfamide cell line lane 11, negative control (no template added). C, Alignment of four deduced OmpP4 sequences among 2 class I strains (35000HP and 82–029362) and 2 class II strains (DMC64 and CIP542). Grey-highlighted residues are conserved within each class but differ between class I and class II strains. Shaded arrows denote the consensus signal peptide cleavage and lipidation site. Construction and characterization of an ompP4mutant We constructed and characterized an isogenic ompP4 mutant of H. ducreyi 35000HP, which was designated 35000HPompP4. PCR amplification of the ompP4 ORF in 35000HPompP4 demonstrated the size shift from 859 bp to 1.7 kb DNA Damage inhibitor expected by addition of the 840 bp kan cassette (Figure 2A). In Southern blotting, the kan probe did not bind to the 35000HP genome but did bind
to an 8.6-kb DNA Selleckchem Bindarit fragment of the mutant genome, as expected. The ompP4 probe bound to a 7.8-kb DNA fragment of the 35000HP genome and to an 8.6-kb fragment of the 35000HPompP4 genome (Figure 2B). Thus, the results from the PCR and Southern blot analyses were consistent with the insertion of a single antibiotic resistance cassette in the appropriate locus for the 35000HPompP4 mutant. Figure 2 Mutagenesis of ompP4 . A, Composite gel of the ompP4 locus amplified using primers that flank the ompP4 ORF. Lane 1, standard; lane 2, 35000HPompP4; lane 3,
35000HP. B, Composite Southern blot of 35000HPompP4 and 35000HP probed with the cloned ompP4 insert (lanes 1, 2) or the kan cassette (lanes 3, 4). Lanes 1 and 4, 35000HPompP4; lanes 2 and 3, 35000HP. C, SDS-PAGE and Coomassie blue staining of OMPs prepared from 35000HPompP4 (lane 2) and 35000HP (lane 3); molecular markers are shown in lane 1, with sizes indicated to the left of the panel. Arrow points to the 30 kDa protein, the predicted size of OmpP4, missing in the ompP4 mutant. Sarkosyl insoluble membrane fractions were prepared from 35000HPompP4 and 35000HP. The fractions obtained from 35000HPompP4 were similar to those of 35000HP, (-)-p-Bromotetramisole Oxalate except for lack of expression of a 30 kDa band (Figure 2C), the predicted size of OmpP4. These data suggest that OmpP4 does sort to the outer membrane . 35000HPompP4 and 35000HP demonstrated similar lipooligosaccharide (LOS) profiles as analyzed by SDS-PAGE (data not shown). 35000HPompP4 and 35000HP demonstrated identical growth rates in broth (data not shown). Role of OmpP4 in experimental human infection Eight healthy adults (three males, five females; 5 Caucasian, 3 black; age range 21 to 56; mean age ± standard deviation, 31 ± 11 years) volunteered for the study.