Nanao Max, Ricard-Blum Sylvie, Di Guilmi Anne Marie, Lemaire David, Lascoux David, Chabert Jacqueline, Attree Ina, Dessen Andréa
Institut de Biologie Structurale (CNRS/CEA/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France.
BMC Microbiol. 2003 Oct 18;3:21. doi: 10.1186/1471-2180-3-21.
Pseudomonas aeruginosa, an increasingly prevalent opportunistic pathogen, utilizes a type III secretion system for injection of toxins into host cells in order to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found both within the bacterial cytoplasm and localized extracellularly, suggesting that it may play more than one role in Pseudomonas infectivity. LcrV, the homolog of PcrV in Yersinia, has been proposed to participate in effector secretion regulation by interacting with LcrG, which may act as a secretion blocker. Although PcrV also recognizes PcrG within the bacterial cytoplasm, the roles played by the two proteins in type III secretion in Pseudomonas may be different from the ones suggested for their Yersinia counterparts.
In this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1-98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content.
PcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia.
铜绿假单胞菌是一种日益普遍的机会致病菌，它利用III型分泌系统将毒素注入宿主细胞以引发感染。该系统的一个关键组分是PcrV，它对细胞毒性至关重要，存在于细菌细胞质内且定位于细胞外，这表明它可能在铜绿假单胞菌的感染性中发挥多种作用。耶尔森菌中PcrV的同源物LcrV已被提出通过与可能作为分泌阻断剂的LcrG相互作用来参与效应蛋白分泌调控。尽管PcrV在细菌细胞质内也能识别PcrG，但这两种蛋白在铜绿假单胞菌III型分泌中所起的作用可能与它们在耶尔森菌中的对应物不同。
在这项研究中，我们通过非变性质谱证明从大肠杆菌表达和纯化的PcrV和PcrG在体外形成1:1复合物。圆二色性结果表明，在没有PcrV的情况下，PcrG高度不稳定；相比之下，单独的PcrV以及PcrV:PcrG复合物都具有高度的结构完整性。表面等离子体共振测量表明，PcrV与PcrG以纳摩尔亲和力（15.6 nM）相互作用且动力学快速，这一观察结果对于PcrG的全长形式（第1至98位氨基酸残基）以及缺少预计具有低二级结构含量的C末端24个残基的形式均有效。
PcrV是铜绿假单胞菌III型分泌系统的关键组分，但它参与毒素分泌的方式尚不清楚。在这里，我们在体外对PcrV和PcrG之间的相互作用进行了表征，并表明PcrG高度不稳定。然而，它通过其前74个氨基酸内的一个区域与PcrV容易结合形成高亲和力复合物。PcrV与一个不稳定分子快速结合和解离这一事实表明PcrV:PcrG复合物具有瞬时性。这些结果与来自pcrV缺失突变体的分析一致，这些分析表明PcrV:PcrG在效应蛋白分泌中可能发挥与耶尔森菌中LcrV:LcrG复合物不同的作用。
