Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria (INTA), Castelar, Buenos Aires, Argentina.
PLoS One. 2010 Feb 4;5(2):e9046. doi: 10.1371/journal.pone.0009046.
Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry.
METHODS/PRINCIPAL FINDINGS: We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled-coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments.
Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis.
肠致病性大肠杆菌（EPEC）和肠出血性大肠杆菌（EHEC）是与人类疾病相关的两种大肠杆菌菌株。这两种病原体的主要毒力因子是表达 III 型分泌系统（TTSS），这使其能够附着在肠道黏膜上，导致特征性的附着和消除病变（A/E）。TTSS 将效应蛋白直接转运到宿主细胞中，从而颠覆了哺乳动物细胞的生物化学过程。
方法/主要发现：我们研究了设计用于抑制 TTSS 的合成肽。CoilA 和 CoilB 肽都代表了转运蛋白 EspA 的卷曲螺旋区，而 CoilD 肽则对应于针状蛋白 EscF 的卷曲螺旋区，这两种肽都能有效抑制 EPEC E2348/69 株依赖 TTSS 的红细胞溶血。CoilA 和 CoilB 肽还减少了同一菌株在 Hep-2 细胞中形成的肌动蛋白足，并损害了 TTSS 介导的蛋白质向上皮细胞的转运。有趣的是，CoilA 和 CoilB 能够阻断 EspA 的组装，从而使 TTSS 不稳定，Tir 易位受阻。CoilA 或 CoilB 肽对 EspA 聚合的阻断作用，也抑制了 EspB 和 EspD 的正确递呈，这可以通过免疫印迹检测到。有趣的是，用 CoilA 肽孵育的细菌的电子显微镜显示 EspA 丝的长度减少。
我们的数据表明，卷曲螺旋肽可以防止 TTSS 装置的组装和功能，这表明这些肽可能是阻断 EPEC 和 EHEC 发病机制的有吸引力的工具。
