Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum - PubMed (original) (raw)

Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum

S K Narayanan et al. Infect Immun. 2001 Sep.

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.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

The leukotoxin locus of F. necrophorum. Boxes, leukotoxin ORF (lktA) and its flanking putative ORFs; lines above boxes, phagemid clones (816, 101, and 611) obtained from the immunoreactive plaques in the cloning experiments; region iPCR, sequence obtained from sequencing a series of inverse PCR clones. Plasmid pSN2000 contains the entire lktA ORF. Below the boxes are the clones expressing the truncated leukotoxin polypeptides. The numbers refer to the nucleotide positions of the boundaries of each truncation relative to the 11,130-bp sequence deposited in GenBank.

FIG. 2

FIG. 2

Kyte-Doolittle hydropathy plots of deduced amino acid sequences from the F. necrophorum leukotoxin gene. Lines above plot, regions of the five truncated LktA polypeptides (BSBSE, SX, GAS, SH, and FINAL).

FIG. 3

FIG. 3

Western blot analysis of truncated forms of purified recombinant leukotoxin protein probed with polyclonal antileukotoxin antiserum (A) and monoclonal antileukotoxin antibody (F7B10 [35]) (B). (C) Western blot of whole-cell lysates from E. coli clones expressing the full-length recombinant leukotoxin probed with the monoclonal antileukotoxin antibody. Abbreviations: MW, molecular weight markers; Lkt, affinity-purified leukotoxin from F. necrophorum; FL-I and FL-UI, full-length clone induced or uninduced, respectively, with IPTG; Super, concentrated F. necrophorum A25 culture supernatant. Arrows, positions of the reactive BSBSE band (B) and the full-length leukotoxin (C). The amount of full-length leukotoxin in the culture supernatant in panel C was insufficient to be visualized as a distinct band in this blot.

FIG. 4

FIG. 4

Evaluation of leukotoxic activity by flow cytometry. Membrane damage was assessed by staining the cells with PI. Shown are the values obtained after counting 10,000 PMNs (stippled bars) or the lymphocyte fraction (hatched bars). Cells were untreated (control) or treated with 200 U of affinity-purified leukotoxin from F. necrophorum (native leukotoxin) or lysates of E. coli harboring expression plasmids bearing the upstream polypeptide (SN200) or the full-length lktA ORF (SN100). U and I, lysates from uninduced cultures and cultures induced with 1 mM IPTG, respectively. Induced lysates were also tested after 1:5, 1:25, and 1:125 dilutions in PBS. The results are the averages of three experiments, and the standard deviations are indicated.

FIG. 5

FIG. 5

Neutralization of the toxicity of the recombinant leukotoxin assessed by flow cytometry. Membrane damage was assessed by staining the cells with PI. Shown are the values obtained after counting 10,000 PMNs (stippled bars) or the lymphocyte fraction (hatched bars). Cells were treated with a lysate obtained from IPTG-induced cultures of E. coli bearing pSN2000. The lysates were untreated (control) or treated with polyclonal antiserum raised against affinity-purified leukotoxin from F. necrophorum (anti-native leukotoxin) or with polyclonal antiserum raised against the recombinant BSBSE and GAS polypeptides. The results are the averages of duplicate experiments, and the standard deviations are indicated.

FIG. 6

FIG. 6

Hybridization patterns of radiolabeled lktA with Southern-blotted _Hae_III-digested restriction fragments of genomic DNAs from F. necrophorum subsp. necrophorum isolates from liver abscesses (lane 1, strain A21; lane 2, A25; lane 3, A39) or ruminal contents (lane 7, RA13; lane 8, RA15; lane 9, RA16; lane 10, RA18; lane 11, RA26; lane 12, RA28; lane 13, RA29) or F. necrophorum subsp. funduliforme isolates from liver abscesses (lane 4, B17; lane 5, B29; lane 6, B35) or ruminal contents (lane 14, RB33; lane 15, RB37). Strains are described in reference . Lane M, DNA molecular weight markers.

Similar articles

Cited by

References

    1. Abhayawardhane Y, Stewart G C. Bacillus subtilis possesses a second determinant with extensive sequence similarity to the Escherichia coli mreB morphogene. J Bacteriol. 1995;177:765–773. - PMC - PubMed
    1. Attwood G T, Klieve A B, Ouwerkerk D, Patel B K. Ammonia-hyperproducing bacteria from New Zealand ruminants. Appl Environ Microbiol. 1998;64:1796–1804. - PMC - PubMed
    1. Baxter-Gabbard K L. A simple method for the large-scale preparation of sucrose gradients. FEBS Lett. 1972;20:117–119. - PubMed
    1. Berg J N, Scanlan C M. Studies of Fusobacterium necrophorum from bovine hepatic abscesses: biotypes, quantitation, virulence, and antibiotic susceptibility. Am J Vet Res. 1982;43:1580–1586. - PubMed
    1. Coyle-Dennis J E, Lauerman L H. Correlation between leukocidin production and virulence of two isolates of Fusobacterium necrophorum. Am J Vet Res. 1979;40:274–276. - PubMed

MeSH terms

Substances

LinkOut - more resources