Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology

Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment, similar to that observed in the sortase mutant. These unanticipated effects suggest that inhibition of sortase by small-molecule inhibitors could similarly lead to the rapid elimination of pathogens from an infected host, making such inhibitors much better anti-bacterial agents than previously believed.细胞壁锚定毒力因子对于革兰氏阳性菌（Gram-positive bacteria）侵染并定殖宿主至关重要。此类蛋白拥有N端引导序列（N-terminal leader sequence）与由LPXTG基序（LPXTG motif）、疏水区域（hydrophobic stretch）以及少量带正电荷氨基酸构成的C端分选信号（C-terminal sorting signal）。分选信号会阻断跨膜转运过程，使分选酶（sortase）得以切割LPXTG基序，进而完成表面锚定。敲除分选酶会阻止毒力因子锚定至细胞壁；然而其对细菌生理的影响尚未得到充分阐释。本研究发现，化脓链球菌（Streptococcus pyogenes）分选酶A（sortase A）的敲除会导致分选中间体的积累，尤其在细胞隔膜处，进而改变细胞形态与生理状态，并破坏膜完整性（membrane integrity）。此类细胞对组织蛋白酶抑菌肽（cathelicidin）高度敏感，并会在血液与血浆中快速被灭杀。上述现象并非因锚定表面蛋白缺失所导致的功能丧失效应，而是由分选中间体的积累特异性引发。降低分选中间体的水平可使分选酶突变株恢复正常形态；而在野生型菌株中表达带有突变LPXTG基序的M蛋白，则会在宿主环境中产生类似分选酶突变株的毒性效应。这些未被预期的结果表明，小分子抑制剂对分选酶的抑制作用同样可促使感染宿主快速清除病原体，使得此类抑制剂成为比此前认知中更为优异的抗菌制剂。