Conservation and diversity of sap homologues and their organization among Campylobacter fetus isolates

Abstract

Campylobacter fetus surface layer proteins (SLPs), encoded by sapA homologues, are important in virulence. In wild-type C. fetus strain 23D, all eight sapA homologues are located in the 54-kb sap island, and SLP expression reflects the position of a unique sapA promoter in relation to the sapA homologues. The extensive homologies in the sap island include both direct and inverted repeats, which allow DNA rearrangements, deletion, or duplication; these elements confer substantial potential for genomic plasticity. To better understand C. fetus sap island diversity and variation mechanisms, we investigated the organization and distribution of sapA homologues among 18 C. fetus strains of different subspecies, serotypes, and origins. For all type A strains, the boundaries of the sap island were relatively consistent. A 187-bp noncoding DNA insertion near the upstream boundary of the sap island was found in two of three reptile strains studied. The sapA homologue profiles were strain specific, and six new sapA homologues were recognized. Several homologues from reptile strains are remarkably conserved in relation to their corresponding mammalian homologues. In total, the observed differences suggest that the sap island has evolved differing genotypes that are plastic, perhaps enabling colonization of varied niches, in addition to antigenic variation.

FIG. 1.

Schematic representation and genomic organization of the sap island and its flanking regions in C. fetus strain 23D. For ease of reading, selected ORFs (02, 09, 20, 31, 44, and A to Ap8) representing Cf0002, Cf0009, Cf0020, Cf0031, Cf0044, and sapA to sapAp8, are labeled, respectively. Each arrow represents the ORF orientation.

FIG. 2.

Identification of the sap island upstream boundary in 18 C. fetus strains. (A) Detection of the presence of Cf0002 (mtfB) by PCR with primers MF and MR. (B) Southern hybridization of Cf0002 with HindIII-digested genomic DNA. The probe is a 707-bp mtfB fragment amplified by PCR from strain 23D. (C) PCR detection of the proximity of Cf0002 to sapA homologues with primers MF and AR (lanes 1 to 11) or primers MF and BR (lanes 12 to 18). (D) Schematic representation of the sap island upstream boundary and the strain 85-388 insertion site and sequence. The black box represents the 5′ conserved regions of the sapA or sapB homologues. The PCR primers and orientations are designated by arrows. (E) PCR amplification of the insertion fragment found between Cf0002 and sapA homologue in strain 85-388 with primers IF and IR.

FIG. 3.

Identification of the sap island downstream boundary. (A) Strategy for detection of Cf0031 location relative to the sap island. The primers are shown as arrows, and the black area indicates the 5′ conserved regions of the sapA or sapB homologues. (B) PCR amplification for Cf0031 in the 18 C. fetus strains with primers DF and DR. (C) PCR identification of the proximity of Cf0031 with sapA or sapB homologues with primers AbF and DR. (D) PCR identification of the proximity of cf0031 with sapA3 with primers 3F1 and DR.

FIG.4.

Analyses of Cf0009, Cf0020, sapC, and sapF locations related to sapA or sapB homologues within the sap island of 18 C. fetus strains. (A) Schematic representation of PCR for Cf0009 analyses, with primers indicated by the arrows; (B) PCR for detection of the presence of Cf0009; (C) Southern hybridization for Cf0009; (D) PCR amplifications for the location of Cf0009 related to the sapA homologues with primer HR paired with AR (for type A strains) or BR (for type B strains); (E) schematic representation of PCR for Cf0020 analysis, with the primers indicated by the arrows; (F) detection of the location of Cf0020 in relation to sapA2 (or sapB2) by PCR with primers 2F and TR; (G) schematic representation of PCR for sapC analysis, with the primers indicated by arrows; (H) PCR for sapC amplification and orientation with primers AR and CR; (I) PCR for sapC analysis with primers BR and CR; (J) schematic representation of PCR for sapF analysis, with the primers indicated by arrows; (K) PCR for sapF amplification and orientation with primers AR or BR and FF.

FIG. 5.

Distribution of eight sapA homologues in 18 C. fetus strains by using two differing sets (I or II) of sap homologue-specific primers (Table 2). The numbers in parentheses indicate the primer locations in the specified genes. The lane numbers representing the strains are the same as in Table 1; lane C represents the no-DNA control.

FIG. 6.

Southern analysis of the distribution of eight sap homologues among C. fetus strains. Each lane contains HindIII-digested genomic DNA from the 18 different C. fetus strains, hybridized with each of eight sap homologue-specific probes and amplified by one set of the PCR products from strain 23D shown in Fig. 5. The numbers in parentheses indicate the probe locations within the specified genes.

FIG.7.

Sequence and antigenicity of sapA homologues identified in the present study. (A) Schematic representation of the structures of new sapA homologues compared to their closest homologues in strain 23D. The different colors indicate regions of sequence identity, the white boxes represent diverse sequences, and the red outlined box in sapA10 represents a deletion in the (gray) semiconserved region compared to sapA4 and sapB9. (B) Immunoblot of recombinant SLPs probed with polyclonal rabbit antiserum to the 97-kDa SLP of type A strain 84-20LP. The SLPs are encoded by sapA8, sapA12, sapB11, sapA13, sapB9, sapA, and sapA10, respectively. An immunoreactive product was observed in each case, except for sapB11. (C) Phylogenetic tree constructed from the nucleotide sequences of 16 sapA or sapB homologues, including the six new homologues. The tree was constructed by using PAUP 4.0bs neighbor-joining method based on Kimura's two-parameter model distance matrices. The size of the deduced or experimentally determined SLP encoded by the homologue is shown at the left. The major branching perfectly conforms to the dichotomy between 97-kDa SLP (hexagonal) and >97-kDa SLP (tetragonal) in crystalline structure (17).