Differential expression of porins OmpP2A and OmpP2B of Haemophilus ducreyi

Abstract

Haemophilus ducreyi is a strict human pathogen and the causative agent of the sexually transmitted disease chancroid. The genome of the human-passaged strain of H. ducreyi (35000HP) contains two homologous genes whose protein products have estimated molecular masses of 46 and 43 kDa. A comparative analysis of the deduced amino acid sequences revealed that these proteins share 27 to 33% identity to the outer membrane protein P2 (OmpP2), a major porin of Haemophilus influenzae. Therefore, these proteins have been designated OmpP2A and OmpP2B, respectively. The detection of ompP2A and ompP2B transcripts by reverse transcriptase PCR indicated that these genes were independently transcribed in H. ducreyi 35000HP. Western blot analysis of outer membrane proteins isolated from a geographically diverse collection of H. ducreyi clinical isolates revealed that OmpP2A and OmpP2B were differentially expressed among these strains. Although PCR analysis suggested that ompP2A and ompP2B were conserved among the strains tested, the differential expression observed was due to nucleotide additions and partial gene deletions. Purified OmpP2A and OmpP2B were isolated under nondenaturing conditions, and subsequent analysis demonstrated that these two proteins exhibited porin activity. OmpP2A and OmpP2B are the first porins described for H. ducreyi.

FIG. 1.

Clustal alignment of the amino acid sequences of OmpP2A and OmpP2B of H. ducreyi 35000HP versus OmpP2 of H. influenzae Rd (typeable). Identical residues are denoted by dark boxes, and similar residues are denoted by light boxes.

FIG. 2.

Genetic organization of the ompP2A and ompP2B loci from H. ducreyi 35000HP. ORF 3 is a putative PulD homolog. Large arrows indicate the direction of transcription. Primers used in RT-PCR are represented by the arrows in panel A, and primers used for cloning are denoted by the arrows in panel B.

FIG. 3.

Detection of ompP2A and ompP2B transcripts by RT-PCR. Total cellular RNA and chromosomal DNA were isolated from H. ducreyi 35000HP. Lanes 1 to 4, 13, and 14, primers 237 and 238, internal to ompP2B; lanes 5 to 8, primers 364 and 365, internal to ompP2A; lanes 9 to 12, primers 400 and 401, spanning the ompP2A-ompP2B intergenic region. Lanes 1, 5, 9, and 13, no nucleic acid template; lanes 2, 6, and 10, no RT; lanes 3, 7, and 11, chromosomal DNA; lanes 4, 8, 12, and 14, total RNA. Lanes marked M indicate molecular size standards in 100-bp increments. Lanes 13 and 14 are controls for RNA integrity.

FIG. 4.

Western blots probed with antisera against rOmpP2A (A) and rOmpP2B (B) as expressed in E. coli BL21(DE3). Lanes 1 and 3, inclusion bodies containing rOmpP2A and rOmpP2B, respectively; lanes 2 and 4, 35000HP OMPs. The asterisk denotes recombinant protein; arrows denote the positions of protein in the OM. Molecular mass (in kilodaltons) is shown on left.

FIG. 5.

Evaluation of OmpP2A and OmpP2B expression by SDS-PAGE (A) and Western blotting (B and C). Lanes: 1, 35000; 2, 35000HP; 3, MF35000; 4, A75; 5, A76; 6, CIP542; 7, HD41; 8, HD151; 9, HD293; 10, HD346; 11, NY8; 12, R3. Corresponding immunoblots were probed with OmpP2A antibodies (B) and OmpP2B antibodies (C). The arrow denotes OmpP2A; the asterisk denotes OmpP2B. Molecular mass (in kilodaltons) is shown on the left.

FIG. 6.

Purification and heat modifiability of native OmpP2A and OmpP2B. Immunoblots were probed with affinity-purified antibodies developed against either OmpP2A (A) or OmpP2B (B). Lanes: 1, unheated; 2, heated; 3, unheated; 4, heated. Heated samples were boiled for 10 min at 100°C.