Identification of a 35-kilodalton serovar-cross-reactive flagellar protein, FlaB, from Leptospira interrogans by N-terminal sequencing, gene cloning, and sequence analysis

Abstract

During the screening of antibodies to pathogenic leptospires, a murine monoclonal antibody (designated M138) was found to react with various serovars. An antigen of approximately 35 kDa from Leptospira interrogans serovar pomona, which reacted strongly with M138, was characterized by N-terminal amino acid sequencing and identified as a flagellin, a class B polypeptide subunit (FlaB) of the periplasmic flagella. The gene encoding the FlaB protein, flaB, was amplified from the genomic DNA of several pathogenic serovars by PCR with a single pair of oligonucleotide primers, suggesting that FlaB is highly conserved among these serovars. Cloning and sequence analysis of flaB from serovar pomona revealed that it contains an 849-bp open reading frame with a G + C content of 46.88% which encodes a 283-amino-acid protein with a calculated molecular mass of 31.297 kDa and a predicted pI of 9.065. A sequence comparison of flagellin proteins revealed that the amino acid sequence is most variable in the central portion of the serovar pomona FlaB, which is believed to contain specific sequence information and which may thus be useful in the design of DNA or synthetic peptide probes suitable for the detection of infection with pathogenic leptospires.