The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence.
The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence.
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DOI：
10.1074/jbc.m116.752782
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发表时间：
2016-12-09
期刊：
影响因子：
--
通讯作者：
Hare SA
中科院分区：
文献类型：
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作者：
Grabe GJ;Zhang Y;Przydacz M;Rolhion N;Yang Y;Pruneda JN;Komander D;Holden DW;Hare SA
许多细菌病原体分泌毒力（效应）蛋白，干扰宿主的免疫信号。SpvD是沙门氏菌的一种效应蛋白，我们先前已经证明它可以负调节NF-κB信号通路并促进沙门氏菌的毒力。肠血清型鼠伤寒沙门氏菌。为了阐明这些观察结果的机制基础，我们确定了SpvD的晶体结构，并表明它采用了木瓜蛋白酶样折叠，具有特征性的半胱氨酸-组氨酸-天冬氨酸催化三联体，包括Cys-73，His-162和Asp-182。SpvD在体外对氨基萤光素连接的肽和蛋白质底物具有体外解偶联活性。C73 A突变废除SpvD活性，表明其功能需要完整的催化三联体。总之，这些结果强烈表明SpvD是一种半胱氨酸蛋白酶。SpvD的氨基酸序列在不同的S.肠杆菌血清型，但位于催化三联体附近的残基161是可变的，鼠伤寒血清型SpvD在该位置具有精氨酸，而肠杆菌血清型甘氨酸。这种变化影响了酶在人工底物上的水解活性，并且可以通过底物对活性位点的可及性来解释。有趣的是，SpvDG 161变体在体外细胞中更有效地抑制NF-κ B介导的免疫应答，并增加鼠伤寒血清型的毒力。总之，我们的研究结果解释了毒力蛋白SpvD的作用的生化基础，并表明单个氨基酸多态性可以影响细菌病原体在其宿主中的整体毒力。
Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising Cys-73, His-162, and Asp-182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro. A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB-mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.