Leukotoxin operon and differential expressions of the leukotoxin gene in bovine Fusobacterium necrophorum subspecies

Liver abscesses in cattle are associated primarily with Fusobacterium necrophorum, a gram negative, pleomorphic, and obligate anaerobe. In cattle, the organism is a normal inhabitant of the rumen and an opportunistic pathogen. There are two subspecies: subsp. necrophorum and subsp. funduliforme. Subspecies necrophorum is more frequently isolated, often in pure culture, from liver abscesses than subspecies funduliforme. Leukotoxin (Lkt), an exotoxin, is a major virulence factor. In subsp. necrophorum, Lkt is a high molecular weight protein that is encoded by a tricistronic leukotoxin operon (lktBAC) and induces apoptosis and necrosis of bovine leukocytes in a dose-dependent manner. The subsp. funduliforme produces lower concentration of leukotoxin and hence less virulent than subsp. necrophorum. The probable cause of low leukotoxin production by subsp. funduliforme is not known. We sequenced the leukotoxin operon and compared it to the operon of subsp. necrophorum. The lkt operon had three genes, lktB, lktA, and lktC and was similar in organization to that of subsp. necrophorum. The subsp. funduliforme LktB and LktA proteins had significant differences in their N-terminal sequences despite high overall amino acid similarities, 87% and 88%, respectively with subsp. necrophorum. The relative expressions of lktA in both subspecies at various growth phases were determined by quantitative PCR (Q-PCR). Data from Q-PCR studies revealed that subsp. funduliforme had a 21.1-fold lower lktA transcript level in mid-log phase cells than subsp. necrophorum. The lktA transcript amounts were lower in all stages of growth in subsp. funduliforme. The maximum concentration of leukotoxin and the highest cytotoxicity on bovine PMNs were observed in the mid-log phase, which corresponded to the highest amount of lktA transcript detected. Therefore, the low toxicity associated with subsp. funduliforme leukotoxin, a less virulent subspecies, may in part be due to the differences in the lktA gene and reduced transcription.