Köller Thomas, Nelson Daniel, Nakata Masanobu, Kreutzer Michael, Fischetti Vincent A, Glocker Michael O, Podbielski Andreas, Kreikemeyer Bernd
Department of Medical Microbiology and Hospital Hygiene, Hospital of Rostock University, Schillingallee 70, Rostock, Germany.
Proteomics. 2008 Jan;8(1):140-8. doi: 10.1002/pmic.200700001.
Streptococcus pyogenes (Spy) (group A streptococci) is an important and exclusively human bacterial pathogen, which uses secreted and surface-associated proteins to circumvent the innate host defense mechanisms and to adhere and internalize into host cells. Thus, investigation of the bacterial extracellular compartments, including secreted and cell wall-associated subproteomes, is crucial for understanding adherence, invasion, and internalization mechanisms as major steps of Spy pathogenesis. Here, we compared a bacteriophage encoded cell wall hydrolase, PlyC, a multimeric lysin of the C1 bacteriophage, with the established glycosidase, mutanolysin, from Streptomyces globisporus for their suitability to efficiently digest Spy cell walls and release cell wall-anchored Spy proteins for subsequent proteome research. Our results show that PlyC is superior for cell wall protein extraction compared to mutanolysin due to its higher activity and specificity as an N-acetylmuramoyl-L-alanine amidase. Furthermore, our experimental design allowed us to delineate the actual localization of the proteins despite contamination with intracellular proteins.
化脓性链球菌（Spy）（A 组链球菌）是一种重要的且仅感染人类的细菌病原体，它利用分泌蛋白和表面相关蛋白来规避宿主的固有防御机制，并粘附和内化到宿主细胞中。因此，研究细菌的细胞外区室，包括分泌的和细胞壁相关的亚蛋白质组，对于理解作为 Spy 发病机制主要步骤的粘附、侵袭和内化机制至关重要。在此，我们将一种噬菌体编码的细胞壁水解酶 PlyC（C1 噬菌体的多聚体溶素）与来自球形链霉菌的已确立的糖苷酶变溶菌素进行比较，以评估它们有效消化 Spy 细胞壁并释放细胞壁锚定的 Spy 蛋白用于后续蛋白质组研究的适用性。我们的结果表明，由于 PlyC 作为 N - 乙酰胞壁酰 - L - 丙氨酸酰胺酶具有更高的活性和特异性，因此与变溶菌素相比，它在提取细胞壁蛋白方面更具优势。此外，我们的实验设计使我们能够在存在细胞内蛋白污染的情况下确定蛋白质的实际定位。
