Allen C A, Adams L G, Ficht T A
Department of Veterinary Pathobiology, Texas A&M University, College Station 77843-4467, USA.
Infect Immun. 1998 Mar;66(3):1008-16. doi: 10.1128/IAI.66.3.1008-1016.1998.
The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential for extra- and intracellular survival in mice.
基于偶然分离出的粗糙变异体生存能力减弱，流产布鲁氏菌的O抗原被描述为主要毒力决定因素。然而，这些突变体缺乏遗传定义以及自然存在的粗糙种绵羊布鲁氏菌和犬布鲁氏菌的毒力，使得解释变得混乱。为了更好地表征O抗原在毒力和生存中的作用，利用转座子诱变产生了在O抗原呈递方面有缺陷的流产布鲁氏菌粗糙突变体。对Tn5插入位点侧翼的DNA进行序列分析，以验证其插入编码脂多糖（LPS）生物合成功能的基因中。不出所料，每个粗糙突变体在小鼠体内的生存能力都减弱了，但观察到突变体之间存在意外差异。为了确定观察到的差异的基础，在体外检查了粗糙LPS的结构以及这些突变体对个体杀伤机制的敏感性。与光滑亲本菌株相比，所有流产布鲁氏菌粗糙突变体对补体介导的裂解的敏感性增加了4至5个对数单位。这些生物体对过氧化氢的敏感性变化不大，这与O抗原无法排除相对较小的分子一致。对多粘菌素B的敏感性，多粘菌素B被用作类似于吞噬细胞中发现的防御素的模型阳离子两亲性肽，揭示了粗糙突变体之间的生存差异，类似于在小鼠中观察到的差异。特别是一个突变体对多粘菌素B表现出超敏感性，并且在小鼠中的生存能力降低。该突变体的特征是粗糙LPS被截断。对该突变体的DNA序列分析揭示了编码磷酸甘露糖变位酶（pmm）的基因中的转座子中断，表明除了O抗原之外，这种活性可能是全长核心多糖合成所必需的。流产布鲁氏菌O抗原似乎对小鼠体内外的生存至关重要。
