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标题：Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways
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作者：Julia L. E. Willett ; Grant C. Gucinski ; Jackson P. Fatherree 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：36
页码：11341-11346
DOI：10.1073/pnas.1512124112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceGram-negative bacteria use contact-dependent growth inhibition (CDI) systems to bind neighboring bacteria and deliver diverse nuclease toxins that inhibit target-cell growth. This process requires toxin transport across the outer and inner membranes of target bacteria to reach DNA and RNA substrates in the cytoplasm. Our data indicate that CDI toxins contain a variable domain that specifies the entry pathway into target bacteria. These "translocation domains" exploit specific integral membrane proteins to deliver linked nuclease domains into the cytoplasm. We also find that CDI translocation domains can be exchanged between CdiA C-terminal toxin domains to deliver nucleases via different routes. These findings reveal a versatile protein-transport mechanism that could potentially be harnessed to deliver other antimicrobial cargoes into Gram-negative bacteria. Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.
关键词：bacterial cell envelope ; DNase activity ; genetic selection ; toxin/immunity genes ; tRNase activity