Characterization of Bartonella clarridgeiae flagellin (FlaA) and detection of antiflagellin antibodies in patients with lymphadenopathy

Abstract

Cat scratch disease (CSD) is a frequent clinical outcome of Bartonella henselae infection in humans. Recently, two case reports indicated Bartonella clarridgeiae as an additional causative agent of CSD. Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir. B. clarridgeiae and B. henselae can be distinguished phenotypically by the presence or absence of flagella, respectively. Separation of the protein content of purified flagella of B. clarridgeiae by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa. N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the flaA gene cloned from B. clarridgeiae. The flagellin of B. clarridgeiae is closely related to flagellins of Bartonella bacilliformis and several Bartonella-related bacteria. Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of B. clarridgeiae are found in patients with CSD or lymphadenopathy. Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls. B. clarridgeiae FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing. Our results further indicate that B. clarridgeiae might be a possible etiologic agent of CSD or lymphadenopathy. However, it remains to be clarified whether antibodies to the FlaA protein of B. clarridgeiae are a useful indicator of acute infection.

FIG. 1

Analysis of B. clarridgeiae flagellin preparation by SDS-PAGE and Coomassie brilliant blue staining. Lanes: MW, molecular mass marker; 1, whole-cell lysates of B. clarridgeiae; 2, cellular debris after shearing off the flagella; 3, crude flagellar protein. The arrow indicates the position of the 41-kDa band identified as the FlaA protein.

FIG. 2

DNA sequence and protein translation of the B. clarridgeiae flagellin locus (flaA). The DNA sequence and translation of the flaA ORF are shown. The putative ribosome binding site (RBS) is underlined, and the N-terminal amino acids determined by protein sequencing are in boldface.

FIG. 3

Comparison of the protein sequence of B. clarridgeiae FlaA with those of other known flagellins. (A) Protein sequence comparison of FlaA from B. clarridgeiae (this work) and B. bacilliformis (accession no. L20677 [sequence corrected by resequencing]) by BLAST search. Identities and similarities are indicated by vertical bars and colons, respectively. (B) Average distance tree of a protein sequence alignment (CLUSTAL W) of flagellins from various bacteria. Abr, Azospirillum brasiliense Laf1 (accession no. U26679); Atu, Agrobacterium tumefaciens FlaD (accession no. U95165); Bba, Bartonella bacilliformis flagellin (accession no. L20677); Bcl, Bartonella clarridgeiae (this work); Bbu, Borrelia burgdorferi flagellar filament 41-kDa core protein (flagellin) (accession no. P11089), Bab, Brucella abortus FliC (accession no. AF019251); Ccr, Caulobacter crescentus flagellin Fljm (accession no. 052529); Cje, Campylobacter jejuni flagellin A (accession no. AAC25643); Eco, Escherichia coli flagellin (accession no. AB028473); Hpy, Helicobacter pylori flagellin B (accession no. AE001449); Lpn, Legionella pneumophila flagellin (accession no. X83232); Pae, Pseudomonas aeruginosa flagellin (accession no. AF034764); Rme, Rhizobium meliloti flagellin flaA (accession no. A39436); Sma, Serratia marcescens flagellin (accession no. D32256); Sty, Salmonella enterica serovar Typhimurium flagellin (accession no. AAB33952); Vch, Vibrio cholerae flagellin (accession no. AF007121); Yen, Yersinia enterocolitica thermoregulated motility protein (accession no. L33468).

FIG. 4

Examination of human sera for antibodies to B. clarridgeiae FlaA protein by immunoblot analysis. Lanes: MW, molecular mass marker; Bc and Fla, amido black-stained B. clarridgeiae whole-cell lysate and flagellin preparation, respectively; 1, positive control (rabbit antiserum); N, negative control without serum; 2, 3, 4, 8, 9, and 10, negative sera; 5, 6, and 7, positive reactive sera containing antiflagellin antibodies. The arrow indicates the position of the 41-kDa FlaA protein.

FIG. 5

Immunoblot analysis of a representative anti-FlaA antibody-positive serum showing the specificity of the anti-B. clarridgeiae-flagellin antibodies. Lanes: MW, molecular mass marker; Bh and Bc, amido black-stained B. henselae and B. clarridgeiae whole-cell antigen, respectively; 1, unabsorbed serum; 2, serum absorbed with whole cells of B. henselae; 3, serum absorbed with whole cells of B. clarridgeiae.