Garcia-Bustos J, Tomasz A
Laboratory of Microbiology, Rockefeller University, New York, New York 10021.
J Bacteriol. 1989 Jan;171(1):114-9. doi: 10.1128/jb.171.1.114-119.1989.
We compared the products of autolytic amidase-catalyzed wall degradation in vivo (in penicillin-induced lysis) and in vitro. Pneumococci labeled in their cell wall stem peptides by radioactive lysine were treated with penicillin, and the nature of wall degradation products released to the medium during lysis of the bacteria was determined. At early times of lysis (20% loss of wall label), virtually all the radioactive peptides released (greater than 94%) were of high molecular size and were still attached to glycan and teichoic acid. At times of more extensive bacterial lysis (56%), progressively larger and larger fractions of the released peptides became free, i.e., detached from glycan and teichoic acid. Analysis of the nondegraded residual wall material by high-resolution high-pressure liquid chromatography revealed that this in vivo-triggered autolysis did not involve selective hydrolysis of some of the chemically distinct stem peptides. Parallel in vitro experiments yielded completely different results. Purified pneumococcal cell walls labeled with radioactive lysine were treated in vitro with low concentrations of pure amidase, and the nature of wall degradation products released during limited hydrolysis and after more extensive degradation was determined. In sharp contrast to the in vivo experiments, the main products of in vitro hydrolysis were free peptides. After a short treatment with amidase (resulting in a 20% loss of label), the material released was enriched for the monomeric stem peptides. At all times of hydrolysis (including the time of extensive degradation), only a relatively small fraction of the released wall peptides was covalently attached to glycan and teichoic acid components (17% as compared with 40% in the intact cell wall). We propose that the in vivo-triggered amidase activity first attacks the amide bonds in some strategically located (or unprotected) stem peptides that hold large segments of cell wall material together. The observations indicate that the in vivo activity of the pneumococcal autolysin is under topographic constraints.
我们比较了自溶酰胺酶在体内（青霉素诱导的裂解过程中）和体外催化细胞壁降解的产物。用放射性赖氨酸标记细胞壁茎肽的肺炎球菌用青霉素处理，然后确定细菌裂解过程中释放到培养基中的细胞壁降解产物的性质。在裂解早期（细胞壁标记损失20%），几乎所有释放的放射性肽（超过94%）都是高分子量的，并且仍然与聚糖和磷壁酸相连。在细菌裂解更广泛时（56%），释放的肽中越来越大比例的肽变得游离，即从聚糖和磷壁酸上脱离。通过高分辨率高压液相色谱对未降解的残留细胞壁物质进行分析表明，这种体内引发的自溶并不涉及对某些化学性质不同的茎肽的选择性水解。平行的体外实验产生了完全不同的结果。用放射性赖氨酸标记的纯化肺炎球菌细胞壁在体外用低浓度的纯酰胺酶处理，然后确定在有限水解和更广泛降解后释放的细胞壁降解产物的性质。与体内实验形成鲜明对比的是，体外水解的主要产物是游离肽。用酰胺酶短暂处理后（导致标记损失20%），释放的物质富含单体茎肽。在所有水解时间（包括广泛降解时），只有相对较小比例的释放的细胞壁肽共价连接到聚糖和磷壁酸成分上（完整细胞壁中为40%，相比之下此处为17%）。我们提出，体内引发的酰胺酶活性首先攻击一些将大部分细胞壁物质连接在一起的位于战略位置（或未受保护）的茎肽中的酰胺键。这些观察结果表明，肺炎球菌自溶素的体内活性受到拓扑结构的限制。
