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Infection and Immunity, September 2001, p. 5447-5455, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5447-5455.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Sanjeev Kumar Narayanan, T. G. Nagaraja, M. M. Chengappa, and George C. Stewart*

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506

Received 1 March 2001/Returned for modification 26 April 2001/Accepted 1 June 2001

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.

* Corresponding author. Mailing address: Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Ave., Manhattan, KS 66506. Phone: (785) 532-4419. Fax: (785) 532-4039. E-mail: stewart{at}vet.ksu.edu.

dagger This paper is contribution no. 01-185-J from the Kansas Agricultural Experiment Station.

Infection and Immunity, September 2001, p. 5447-5455, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5447-5455.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Tadepalli, S., Stewart, G. C., Nagaraja, T. G., Narayanan, S. K. (2008). Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity. J Med Microbiol 57: 225-231 [Abstract] [Full Text]  
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