Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum

Abstract

Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

Genome Sequence and Analysis of the Oral Bacterium Fusobacterium nucleatum Strain ATCC 25586

“…The functions of these are not known. A major virulence factor, a leukotoxin (LktA) of F. necrophorum (30), is absent from F. nucleatum.…”

Liver abscesses in cattle: A review of incidence in Holsteins and of bacteriology and vaccine approaches to control in feedlot cattle12

“…necrophorum produces more leukotoxin than funduliforme (Coyle-Dennis and Lauerman, 1979;Emery et al, 1985;Tan et al, 1992), and isolates from liver abscesses tend to be more leukotoxic than isolates from the rumen, indicating a selective advantage for high-leukotoxin-producing strains to survive in the ruminal epithelium and in the liver parenchymal tissue (Tan et al, 1994). Leukotoxin is encoded by a gene designated as lktA, which is the second gene in a 3-gene operon of lktB, lktA, and lktC (Narayanan et al, 2001). The lktA leukotoxin gene ORF is 9726 bp, encoding a protein of 3,241 amino acids with a molecular weight of 336,956 da.…”

“…Narayanan et al (2003) evaluated the efficacy of recombinant leukotoxin of F. necrophorum in a mouse model for liver abscesses. The gene encoding the leukotoxin (lktA) in F. necrophorum was cloned, sequenced, and expressed in E. coli (Narayanan et al, 2001). Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, 5 overlapping leukotoxin gene truncations were constructed.…”

“…Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, 5 overlapping leukotoxin gene truncations were constructed. Five recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by Ni-affinity chromatography (Narayanan et al, 2001). All polypeptides were immunogenic; 2 polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection.…”

Fusobacterium necrophorumLeukotoxin Induces Activation and Apoptosis of Bovine Leukocytes

“…Apoptosis has been reported in target cells exposed to leukotoxins from P. haemolytica and A. actinomycetemcomitans (20,21,40,42,47). The nucleotide sequence encoding F. necrophorum leukotoxin and the deduced amino acid sequence suggested that the leukotoxin is a novel protein unrelated to any known leukotoxins or other bacterial exotoxins (29). Therefore, the mode of action for F. necrophorum leukotoxin is of interest.…”