Tadepalli Sambasivarao, Stewart George C, Nagaraja T G, Narayanan Sanjeev K
Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506-5606, USA.
Anaerobe. 2008 Feb;14(1):13-8. doi: 10.1016/j.anaerobe.2007.09.001. Epub 2007 Oct 4.
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.
牛的肝脓肿主要与坏死梭杆菌有关，这是一种革兰氏阴性、多形态的专性厌氧菌。在牛体内，该菌是瘤胃的正常寄居菌，也是一种机会致病菌。它有两个亚种：坏死亚种和底形亚种。与底形亚种相比，坏死亚种更常从肝脓肿中分离出来，且常为纯培养物。白细胞毒素（Lkt）是一种外毒素，是主要的毒力因子。在坏死亚种中，Lkt是一种高分子量蛋白质，由一个三顺反子白细胞毒素操纵子（lktBAC）编码，并以剂量依赖的方式诱导牛白细胞凋亡和坏死。底形亚种产生的白细胞毒素浓度较低，因此其毒力低于坏死亚种。底形亚种白细胞毒素产生量低的可能原因尚不清楚。我们对白细胞毒素操纵子进行了测序，并将其与坏死亚种的操纵子进行了比较。lkt操纵子有三个基因，lktB、lktA和lktC，其结构与坏死亚种的相似。尽管底形亚种的LktB和LktA蛋白与坏死亚种的总体氨基酸相似性分别高达87%和88%，但其N端序列存在显著差异。通过定量PCR（Q-PCR）测定了两个亚种在不同生长阶段lktA的相对表达量。Q-PCR研究数据显示，在对数中期细胞中，底形亚种的lktA转录水平比坏死亚种低21.1倍。在底形亚种的所有生长阶段，lktA转录量都较低。在对数中期观察到白细胞毒素的最大浓度以及对牛多形核白细胞的最高细胞毒性，这与检测到的lktA转录量最高相对应。因此，毒力较低的底形亚种白细胞毒素的低毒性可能部分归因于lktA基因的差异和转录减少。
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