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Holin triggering in real time

机译：Holin实时触发

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摘要

During X infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105-GFP chimera and real-time decon-volution fluorescence microscopy to show that the S105-GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an early-lysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105_(ΔTMD1)-mcherryfp, in the product of which the S105 sequence deleted for the first trans-membrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105-GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105-GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis.
机译：在X感染期间，Holin S105无害地积聚在膜中，直到在等位基因特定的时间突然触发形成史无前例的大小（> 300 nm）的不规则孔，从细胞质中释放出内溶素，从而在几秒钟内裂解。在这里，我们使用功能性S105-GFP嵌合体和实时解卷积荧光显微镜技术显示，S105-GFP融合体以均匀分布的方式积累，直到在1分钟内突然形成聚集物或筏。致命的触发。此外，与非致死性S105突变体的同基因融合仍保持均匀分布，而与早期裂解的突变​​体的融合显示出早期触发和早期筏形成。 WT，早期和非致死等位基因的蛋白质积累速率相同。光漂白后的荧光恢复（FRAP）表明，非致死突变体和未触发的WT杂种在膜中具有很高的流动性，而WT筏基本上是不动的。最后，将抗胆碱等位基因S105_（ΔTMD1）-mcherryfp与mCherryFP融合，在该产物中，第一个跨膜结构域缺失的S105序列。该杂种保留了全部的抗胆红素活性，因为它阻止了S105-GFP融合产生的致死孔，在整个宿主膜上均匀积累，并阻止了S105-GFP蛋白形成筏。这些发现表明，当holin达到临界浓度并成核形成筏时，噬菌体裂解发生，类似于在嗜盐细菌中诱导细菌视紫红质后紫色膜形成的开始。霍林功能的这种模型可能与哺乳动物细胞中的过程有关，包括非包膜病毒的释放和细胞凋亡。

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  • 作者

    Rebecca White; Shinobu Chiba; Ting Pang; Jill S. Dewey; Christos G. Savva; Andreas Holzenburg; Kit Pogliano; Ry Young;

  • 作者单位

    Department of Biology,Texas A&M University, College Station, TX 77843;

    Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0377;

    Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843;

    Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843;

    The Microscopy and Imaging Center, Texas A&M University, College Station, TX 77843;

    Department of Biology,Texas A&M University, College Station, TX 77843,The Microscopy and Imaging Center, Texas A&M University, College Station, TX 77843;

    Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0377;

    Department of Biology,Texas A&M University, College Station, TX 77843;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    bacteriophage; latent period; peptide linker;

    机译：噬菌体潜伏期肽接头;

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