Raz Assaf, Fischetti Vincent A
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18549-54. doi: 10.1073/pnas.0808301105. Epub 2008 Nov 18.
Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many gram-positive bacteria. The attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved C-terminal LPXTG motif and covalently attaches the protein to the peptidoglycan precursor lipid II. It is unclear how the sorting reaction is regulated spatially and what part sortase localization plays in determining the distribution of surface proteins. This is mainly the result of inadequate immunofluorescence techniques required to resolve these issues in certain bacterial pathogens. Here we describe the utilization of the phage lysin PlyC to permeabilize the cell wall of Streptococcus pyogenes to antibodies, thereby allowing the localization of sortase A using deconvolution immunofluorescence microscopy. We find that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M protein anchoring. Sortase distribution to the new septum begins at a very early stage, culminates during septation, and decays after division is completed. This implies that the sorting reaction is a dynamic, highly regulated process, intimately associated with cell division. The ability to study cytoplasmic and membrane antigens using deconvolution immunofluorescence microscopy will facilitate further study of cellular processes in S. pyogenes.
细胞壁肽聚糖锚定的表面蛋白是许多革兰氏阳性菌中至关重要的毒力因子。这些蛋白通过转肽反应附着于肽聚糖，在此过程中，分选酶切割保守的C末端LPXTG基序，并将蛋白共价连接到肽聚糖前体脂质II上。目前尚不清楚分选反应在空间上是如何调控的，以及分选酶定位在决定表面蛋白分布中发挥何种作用。这主要是由于在某些细菌病原体中解决这些问题所需的免疫荧光技术不足。在此，我们描述了利用噬菌体溶素PlyC使化脓性链球菌细胞壁对抗体通透，从而通过去卷积免疫荧光显微镜对分选酶A进行定位。我们发现分选酶定位于不同的膜性病灶内，其中大多数与分裂隔膜相关，并与活性M蛋白锚定区域共定位。分选酶向新隔膜的分布在很早阶段就开始，在隔膜形成过程中达到顶峰，并在分裂完成后衰减。这意味着分选反应是一个动态的、高度调控的过程，与细胞分裂密切相关。利用去卷积免疫荧光显微镜研究细胞质和膜抗原的能力将有助于进一步研究化脓性链球菌中的细胞过程。
