Godoy V G, Dallas M M, Russo T A, Malamy M H
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Infect Immun. 1993 Oct;61(10):4415-26. doi: 10.1128/iai.61.10.4415-4426.1993.
Two Bacteroides fragilis neuraminidase-deficient mutants were used to study the role of neuraminidase activity in growth of B. fragilis in tissue culture monolayers (CHO cells) and in the in vivo rat granuloma pouch. The nanH structural gene for neuraminidase was cloned from B. fragilis TM4000 and was used to create two isogenic strains with chromosomal disruptions at the nanH gene. B. fragilis VRC404 contains an insertion flanked by disrupted copies of the nanH gene, and B. fragilis VRC426 contains a deletion of a significant portion of nanH coding sequences. The insertion mutant VRC404 is capable of reverting to nanH+. It grew as well as the wild type in CHO monolayers. However, between 48 and 72 h after infection, the bacterial population was enriched with nanH+ bacterial cells (10 to 20%). In the rat pouch 48 h after infection, more than 90% of the population sampled had become nanH+. The deletion mutant VRC426 showed a severe growth defect in the rat pouch model. In addition, VRC426 was efficiently outgrown by the wild type in competition experiments, even when the mutant was present at 10 times the number of wild-type cells at the time of infection. A common characteristic of both model systems is a drastic decrease in the free glucose concentration 16 to 24 h postinfection. We suggest that neuraminidase activity may be required for B. fragilis to grow to maximal levels in the tissue culture and rat pouch systems by making other carbon sources available after glucose levels are reduced.
使用两株脆弱拟杆菌神经氨酸酶缺陷型突变体来研究神经氨酸酶活性在脆弱拟杆菌于组织培养单层细胞（CHO细胞）及体内大鼠肉芽肿袋中的生长过程中的作用。从脆弱拟杆菌TM4000中克隆出神经氨酸酶的nanH结构基因，并用于构建两株在nanH基因处发生染色体破坏的同基因菌株。脆弱拟杆菌VRC404含有一个两侧为nanH基因破坏拷贝的插入片段，而脆弱拟杆菌VRC426含有nanH编码序列的一大段缺失。插入突变体VRC404能够回复为nanH+。它在CHO单层细胞中的生长情况与野生型相同。然而，在感染后48至72小时之间，细菌群体中富含nanH+细菌细胞（10%至20%）。在感染后48小时的大鼠袋中，所采样群体中超过90%已变为nanH+。缺失突变体VRC426在大鼠袋模型中表现出严重的生长缺陷。此外，在竞争实验中，即使突变体在感染时的数量是野生型细胞的10倍，VRC426仍被野生型有效地超越。两个模型系统的一个共同特征是感染后16至24小时游离葡萄糖浓度急剧下降。我们认为，神经氨酸酶活性可能是脆弱拟杆菌在组织培养和大鼠袋系统中生长至最大水平所必需的，因为在葡萄糖水平降低后，它能使其他碳源变得可用。
