Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Environmental Medicine and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Microbiology (Reading). 2012 Aug;158(Pt 8):2133-2143. doi: 10.1099/mic.0.057273-0. Epub 2012 May 24.
Streptococcus mutans, a causative agent of dental caries in humans, adapts to changing environmental conditions, such as pH, in order to survive and cause disease in the oral cavity. Previously, we have shown that S. mutans increases the proportion of monounsaturated membrane fatty acids as part of its acid-adaptive strategy. Membrane lipids function as carriers of membrane fatty acids and therefore it was hypothesized that lipid backbones themselves could participate in the acid adaptation process. Lipids have been shown to protect other bacterial species from rapid changes in their environment, such as shifts in osmolality and the need for long-term survival. In the present study, we have determined the contribution of cardiolipin (CL) to acid resistance in S. mutans. Two ORFs have been identified in the S. mutans genome that encode presumptive synthetic enzymes for the acidic phospholipids: phosphatidylglycerol (PG) synthase (pgsA, SMU.2151c) and CL synthase (cls, SMU.988), which is responsible for condensing two molecules of PG to create CL. A deletion mutant of the presumptive cls gene was created using PCR-mediated cloning; however, attempts to delete pgsA were unsuccessful, indicating that pgsA may be essential. Loss of the presumptive cls gene resulted in the inability of the mutant strain to produce CL, indicating that SMU.988 encodes CL synthase. The defect in cls rendered the mutant acid sensitive, indicating that CL is required for acid adaptation in S. mutans. Addition of exogenous CL to the mutant strain alleviated acid sensitivity. MS indicated that S. mutans could assimilate exogenous CL into the membrane, halting endogenous CL incorporation. This phenomenon was not due to repression, as a cls gene transcriptional reporter fusion exhibited elevated activity when cells were supplemented with exogenous CL. Lipid analysis, via MS, indicated that CL is a reservoir for monounsaturated fatty acids in S. mutans. We demonstrated that the cls mutant exhibits elevated F-ATPase activity but it is nevertheless unable to maintain the normal membrane proton gradient, indicating cytoplasmic acidification. We conclude that the control of lipid backbone synthesis is part of the acid-adaptive repertoire of S. mutans.
变形链球菌是导致人类龋齿的病原体，它能适应口腔内 pH 值等环境变化，从而生存并引发疾病。此前，我们已证实变形链球菌会增加单不饱和膜脂肪酸的比例，以此作为其适应酸性环境的策略之一。膜脂作为膜脂肪酸的载体，因此我们推测脂质主链本身可能参与了酸适应过程。脂质已被证明可保护其他细菌免受渗透压快速变化和长期生存等环境变化的影响。在本研究中，我们确定了心磷脂（CL）对变形链球菌耐酸性的贡献。在变形链球菌基因组中已鉴定出两个 ORF，它们分别编码酸性磷脂的假定合成酶：磷脂酰甘油（PG）合酶（pgsA，SMU.2151c）和 CL 合酶（cls，SMU.988），后者负责将两个 PG 分子缩合以产生 CL。使用 PCR 介导的克隆技术创建了推定 cls 基因的缺失突变体；然而，删除 pgsA 的尝试均未成功，表明 pgsA 可能是必需的。推定 cls 基因的缺失导致突变株无法产生 CL，表明 SMU.988 编码 CL 合酶。cls 缺陷使突变株对酸敏感，表明 CL 是变形链球菌适应酸性的必需条件。向突变株中添加外源性 CL 可减轻其酸性敏感性。MS 表明，变形链球菌可以将外源性 CL 吸收到膜中，从而阻止内源性 CL 的掺入。这种现象不是由于抑制引起的，因为当细胞补充外源性 CL 时，cls 基因转录报告融合体表现出更高的活性。通过 MS 进行的脂质分析表明，CL 是变形链球菌中单不饱和脂肪酸的储备库。我们证明 cls 突变体表现出升高的 F-ATPase 活性，但它仍然无法维持正常的膜质子梯度，表明细胞质酸化。我们得出结论，脂质主链合成的控制是变形链球菌酸适应机制的一部分。
