The Bartonella vinsonii subsp. arupensis immunodominant surface antigen BrpA gene, encoding a 382-kilodalton protein composed of repetitive sequences, is a member of a multigene family conserved among bartonella species

Abstract

Bartonella proteins that elicit an antibody response during an infection are poorly defined; therefore, to characterize antigens recognized by the host, a Bartonella genomic expression library was screened with serum from an infected mouse. This process led to the discovery of a Bartonella vinsonii subsp. arupensis gene encoding a 382-kDa protein, part of a gene family encoding large proteins, each containing multiple regions of repetitive segments. The genes were termed brpA to -C (bartonella repeat protein) and bore significant similarity to genes encoding the BadA adhesin protein and members of the variably expressed outer membrane protein family of proteins from Bartonella henselae and Bartonella quintana, respectively.

FIG. 1.

Flow chart of steps describing the identification of the initial 6.1 clone through the DNA sequencing of the 26-kb Bva-Brp fosmid clone. (1) Bartonella strain na19103nm genomic lambda library screened with antiserum from a mouse infected with Bartonella strains isolated from Peromyscus and Spermophilus reveals several positive plaques. A positive clone, 6.1, was selected for further characterization. (2) Western blot of the 58-kDa recombinant protein expressed by clone 6.1 reacted with the antiserum used in the library screening in step 1. Lane A, Escherichia coli with expression vector with no insert; lane B, E. coli with the 6.1 cloned insert. (3) The 1.5-kb insert of 6.1 is sequenced. Repeat regions are recognized. (4) PCR primers are synthesized from the 6.1 termini and used for amplification from Bartonella species genomic DNA. A similar-sized amplicon is observed from B. vinsonii subsp. arupensis (lane d). Lanes: a, B. quintana; b, B. henselae; c, B. vinsonii subsp. berkhoffii; d, B. vinsonii subsp. arupensis; e, B. elizabethae; f, B. bacilliformis; g, strain na19103 nm; h, clone 6.1. (5) B. vinsonii subsp. arupensis fosmid library is hybridized with the clone 6.1 probe to identify large inserts harboring analogous region. Positive clones are selected for analysis. (6) A B. vinsonii subsp. arupensis fosmid clone containing a 26-kb insert, Bva-Brp, was sequenced and analyzed.

FIG. 2.

Southern blot of Bartonella spp. genomic DNA. Genomic DNA samples were digested with either BamHI or XbaI as indicated and hybridized to the clone Bva-6.1 probe (for the location within the gene, see Fig. 3A). Bh = B. henselae, Bq = B. quintana, Be = B. elizabethae, Bvb = B. vinsonii subsp. berkhoffii, Bva = B. vinsonii subsp. arupensis, Bb = B. bacilliformis. Molecular weight markers in kb are noted on the left. A 23- to 25-kb weakly hybridizing band for B. quintana is present.

FIG. 3.

Organization of genes on the 26-kb Bva-Brp clone of B. vinsonii subsp. arupensis. (A) brpA, brpB, and brpC coding sequences are represented by rectangular bars, with the arrows showing the direction of transcription. The hatched bars within the coding sequences denote portions of the genes composed of repetitive sequences. Genes downstream of brpA are abbreviated as HP (hypothetical protein), EPP (exopolyphosphatase), ftsJ (cell division protein), and GBP (GTP-binding protein). The 409-bp intervening sequence between the stop codon of brpC and the start codon of brpB is shown. The bidirectional arrows indicate inverted repeats characteristic of transcriptional termination sites for brpC. Two putative brpB promoter sites based on consensus sequences for the ribosome binding site (RBS) (underlined), the −10 site (underlined with a dot-and-dash line), and the −35 site (heavily underlined line) are denoted. The region immediately downstream of the stop codon for brpB is shown with inverted repeats illustrated with the bidirectional arrows. The immediate upstream noncoding region is shown for brpA with putative RBS, −10, and −35 sites. The location of the original truncated Bva-6.1 clone that generated the recombinant protein used in this study is shown above brpA. A scale in kbp is above the diagram. (B) Signal peptides deduced from the amino acid sequences for brpA and brpB. Charged residues are italicized, the hydrophobic central region is in bold, and putative SPase I cleavage sites are underlined. Cleavage sites predicted by the SignalP program follow the underlined VNA and VVA of BrpA and BrpB, respectively.

FIG. 4.

Dot plot matrix analysis at the nucleotide level of the 26-kb Bva-Brp insert plotted against itself. (A) Analysis performed at an 80% homology level. Each line represents an area with a corresponding homologous area with an identity level of at least 80%. The bold diagonal in the center represents the 100% match of the sequence with itself. Areas illustrating the locations of brpA, brpB, and brpC are denoted along the diagonal. The boxed regions of the matrix indicate homologous segments to brpA within brpA, brpB, and brpC. (B) Same as panel A, except the analysis was performed at the 70% identity level, which illustrates the areas of homology within and among the genes more clearly. Numbers on the x and y axes denote the nucleotide positions of the 26-kb Bva-Brp cloned insert.

FIG. 5.

(A) Dot plot analysis at the amino acid level of BrpA plotted against itself at the 70% identity level. The diagonal in the center indicates the 100% match of the sequence with itself. Circles surround the largest repeated regions corresponding to amino acid positions 1279 to 1504, 1800 to 2022, 2504 to 2738, and 2825 to 3046. Numbers on the x and y axes denote the amino acid positions for BrpA. (B) Amino acid alignment of repeats 1279 to 1504, 1800 to 2022, 2504 to 2738, and 2825 to 3046. Residues in bold italics represent divergence from the consensus.

FIG. 6.

(A) Immunoblot of whole-cell Bartonella lysates with anti-Bva 6.1. Anti-Bva-6.1 was used at a 1:1,000 dilution. Proteins were fractionated on a 7.5% polyacrylamide gel. Molecular size markers (in kilobases) are noted at the left. (B) Recombinant Bva-6.1 protein blotted against antisera from mice infected with rodent Bartonella strains. Sources of antibodies are indicated. Antibodies were used at a 1:500 dilutin. The lower reacting bands in the anti-Bva-6.1 blot are breakdown products of recombinant BrpA. (C) Immunoblot of recombinant Bva-6.1 protein blotted with polyclonal antisera generated against Bartonella sp. whole-cell lysates. Antibodies were used at a 1:500 dilution. Bh = B. henselae, Bq = B. quintana, Bb = B. bacilliformis, Be = B. elizabethae, Bvv = B. vinsonii subsp. vinsonii.

FIG. 7.

Immunofluorescent staining of Bartonella cells. (A) B. vinsonii subsp. arupensis stained with anti-Bva 6.1; (B) B. quintana stained with anti-Bva 6.1; (C) B. vinsonii subsp. arupensis stained with preimmunized mouse serum. Arrows in panel A point to the concentrated staining in the polar regions of the cells. Magnification, ×1,000.