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。此前，我们在加赛乳杆菌JCM1131T中发现了对胆酸的适应，胆酸是人体肠道中主要的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的致死浓度下。在15 mM CA作用7h后，适应细胞的活力没有明显下降，而非适应细胞在10~(-3)~(-4)的倍数下迅速丧失活力。在适应细胞中，CA的存在明显减轻了细胞膜的损伤，这表明细胞膜中的脂质成分发生了变化。从适应细胞和非适应细胞中提取的总脂被分成三个组分，中性脂、糖脂和磷脂。每一组分的含量测定显示没有显著的…适应细胞和非适应细胞之间有更多的蚂蚁变化。同样，各组分的脂肪酸组成也没有显著差异。然而，糖脂和磷脂的组成发生了显著的变化。在糖脂中，糖链长度在适应过程中被检测到增加。此外，在磷脂组分中观察到心磷脂(CL)的相对含量显著增加。为了评价CL在CA适应中的生理意义，进行了囊泡实验。发现CL对脂质体具有抵抗CA攻击的作用，提示CL在适应机制中起着关键作用。然后，我们筛选了两个可能的心磷脂合成酶基因(CLS)单独或联合缺失的突变体，并研究了它们在CA适应中的能力。虽然两个单基因敲除突变体的CL水平都低于野生型JCM1131T，但在4 mM CA暴露后，CL水平升高，并且两个突变体对野生型菌株具有相似的CA适应性。该双基因敲除突变体的CL含量可忽略不计，但仍表现出CA适应，说明CL在CA适应中的作用是可有可无的。然而，与野生型菌株相比，该突变体的糖脂组分显著降低，磷脂组分显著增加，并且这些变化在CA适应后更加显著。因此，CL被发现是细菌细胞膜脂质组成的关键决定因素。较少

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