Survival strategy of intestinal lactic acid bacteria in the gut : functional analysis of cell surface structure involved in bile acid adaptation

游离胆汁酸（FBAs）被认为是最有害的应激化合物，通过其膜破坏作用抑制肠道细菌的生长。在此之前，我们发现乳酸杆菌JCM1131T对胆酸（CA）具有适应性，胆酸是人类肠道中主要的FBAs之一。在这种适应中，指数生长的细胞首先暴露在4 mM的亚致死浓度的CA中30分钟，然后暴露在15 mM的致死浓度中。在15 mM CA存在下，适应的细胞在7小时后也没有表现出明显的活力下降，而未适应的细胞则以10-3~-4的因子迅速失去活力。在适应的细胞中，在CA的存在下观察到膜损伤的显著缓解，这表明细胞包膜中的脂质组成发生了变化。从适应细胞和非适应细胞中提取的总脂质分为中性脂质、糖脂质和磷脂三部分。在适应细胞和非适应细胞之间，蚁群的变化更大。同样，在脂肪酸组成中也没有发现显著差异。然而，在糖脂和磷脂组成中检测到显著的变化。在糖脂中，在适应过程中检测到糖链长度的增加。此外，磷脂组分中心磷脂（CL）的相对含量显著增加。为了评估CL在CA适应中的生理重要性，我们进行了囊泡实验。研究发现，脂质体对CA的攻击具有抗性，提示脂质体在适应机制中起关键作用。然后获得了两个假定的心磷脂合成酶基因（cls）单独或组合缺失的突变体，并研究了它们对CA的适应能力。虽然这两个单敲除突变体的CL水平都低于野生型JCM1131T，但暴露于4 mM CA后发现CL水平升高，并且在这两个突变体中观察到与野生型菌株相似的CA适应。双敲除突变体的CL含量可以忽略不计，但仍具有CA适应能力，表明CL在CA适应中的作用是可以忽略的。然而，在该突变体中，与野生型菌株相比，糖脂含量显著降低，磷脂含量显著增加，这些变化在CA适应后更为明显。因此，发现CL是细菌细胞膜脂质组成的关键决定因素。少

Free bile acids (FBAs) are considered the most deleterious stress compounds that inhibit the growth of intestinal bacteria by their membrane-damaging effect. Previously, we found adaptation in Lactobacillus gasseri JCM1131T to cholic acid (CA), one of the major FBAs in human intestine. In this adaptation, exponentially growing cells were first exposed to 4 mM CA, a sub-lethal concentration, for 30 min and then to lethal concentration at 15 mM. The adapted cells did not show any appreciable decrease in viability in the presence of 15 mM CA even after 7 h, while non-adapted cells rapidly lost viability by the factors of 10-3~-4. In the adapted cells a significant relief of membrane damage was observed in the presence of CA, suggesting alterations in lipid composition in the cell envelope.Total lipids extracted from adapted and non-adapted cells were separated into three fractions, neutral lipids, glycolipids and phospholipids. Determination of amount of each fraction revealed no signific … More ant changes between adapted and non-adapted cells. Likewise, significant differences were neither found in fatty acids compositions of each fraction. However, significant alterations were detected in glycolipids and phospholipids compositions. In glycolipids, increases in the length of sugar chain were detected during the adaptation. Furthermore, a significant increase in relative amount of cardiolipin (CL) was observed in the phospholipid fraction.To evaluate physiological importance of CL in CA adaptation, vesicle experiment was conducted. It was found that CL confers resistance to the liposome against CA attack, suggesting a pivotal role of CL in adaptation mechanism.Mutants having deletion in two putative cardiolipin synthase genes (cls) singly or in combination were then derived, and their abilities in CA adaptation were investigated. Although CL levels of the both single-knockout mutants were lower than that of the wild type JCM1131T, the levels were found to increase after exposure to 4 mM CA, and a comparable CA adaptation to the wild type strain was observed in both the mutants. The double-knockout mutant possessed negligible amount of CL but still showed CA adaptation, suggesting a dispensable role of CL in CA adaptation. However, in this mutant, significant decrease in glycolipid fraction and significant increase in phospholipid fraction were observed as compared to wild-type strain and these shifts were more prominent after CA adaptation. Therefore, CL was found to work as a key determinant for the lipid composition of the bacterial cell membrane. Less