Abstract：
The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, β-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and β-dystrobrevin. The interaction between SseM and β-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of β-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence.
OBJECTIVE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein β-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.
OBJECTIVE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein β-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.
摘要：
沙门氏菌致病性岛2（SPI-2）编码的注射体被认为易位28个效应子。这里,我们使用蛋白质组学方法描述了一种侵袭性非伤寒沙门氏菌肠炎的临床菌株的分泌组特征,该菌株已突变以引起SPI-2注射体效应物的过度分泌.连同许多已知的效应器，我们发现了新的SseM蛋白.sseM广泛分布在肠沙门氏菌的五个亚种中，在许多临床相关的血清型中发现，并与SPI-2效应基因pipB2共转录。SseM的易位需要功能性SPI-2注射剂。在人类细胞中表达后，SseM与肌营养不良蛋白相关蛋白复合物（DAPC）的五个成分相互作用，即,β-2-syntrophin,肌萎缩蛋白/肌萎缩蛋白，α-catulin,α-赤霉素,和β-赤霉素。在鼠伤寒沙门氏菌感染的细胞中验证了SseM与β-2-syntrophin和α-dystrobrevin之间的相互作用，并依赖于β-2-syntrophin的突触后密度-95/椎间盘大/zonula闭塞-1（PDZ）结构域和对应于SseM中PDZ结合基序（PBM）的序列。在全身性疾病的鼠伤寒沙门氏菌/小鼠模型中，ΔsseM突变菌株与野生型菌株相比具有较小的竞争优势。该表型由表达来自鼠伤寒沙门氏菌或肠炎沙门氏菌的野生型SseM的质粒补充,并且依赖于SseM的PBM。因此,沙门氏菌效应物内的PBM介导与DAPC的相互作用并调节小鼠中细菌的全身生长。此外，ΔsseM突变株在骨髓源性巨噬细胞中表现出增强的复制，证明SseM抑制细胞内细菌生长以调节沙门氏菌毒力。
目标：在肠沙门氏菌中，由沙门氏菌致病性岛2（SPI-2）编码的注射体机制在五个亚种之间是保守的，并将蛋白质（效应物）递送到宿主细胞中，这是沙门氏菌毒力所必需的。SPI-2注射效应物的鉴定和功能表征促进了我们对沙门氏菌及其宿主之间相互作用的理解。使用制备分泌蛋白的优化方法和侵袭性非伤寒沙门氏菌肠血清肠炎菌株D24359的临床分离株，我们鉴定了22种已知的SPI-2注射小体效应子和一种新的效应子-SseM。SseM在鼠感染期间调节细菌生长，并具有与突触后密度95/盘大/小带闭塞1（PDZ）结合基序相对应的序列，该基序对于与含PDZ的宿主蛋白β-2-syntrophin和肌营养不良蛋白相关蛋白复合物（DAPC）的其他成分相互作用至关重要。据我们所知,SseM在沙门氏菌效应物中是独特的，它含有功能性的PDZ结合基序，并且是第一个靶向DAPC的细菌蛋白。
目标：在肠沙门氏菌中，由沙门氏菌致病性岛2（SPI-2）编码的注射体机制在五个亚种之间是保守的，并将蛋白质（效应物）递送到宿主细胞中，这是沙门氏菌毒力所必需的。SPI-2注射效应物的鉴定和功能表征促进了我们对沙门氏菌及其宿主之间相互作用的理解。使用制备分泌蛋白的优化方法和侵袭性非伤寒沙门氏菌肠血清肠炎菌株D24359的临床分离株，我们鉴定了22种已知的SPI-2注射小体效应子和一种新的效应子-SseM。SseM在鼠感染期间调节细菌生长，并具有与突触后密度95/盘大/小带闭塞1（PDZ）结合基序相对应的序列，该基序对于与含PDZ的宿主蛋白β-2-syntrophin和肌营养不良蛋白相关蛋白复合物（DAPC）的其他成分相互作用至关重要。据我们所知,SseM在沙门氏菌效应物中是独特的，它含有功能性的PDZ结合基序，并且是第一个靶向DAPC的细菌蛋白。
