巨大的食细菌病毒弥合了生命与非生命之间的鸿沟 诸平 Fig. 1 Depiction of huge phages (red, left) and normal phages infecting a bacterial cell. The huge phage injects its DNA into the host cell, where Cas proteins -- part of the CRISPR immune system typically found only in bacteria and archaea -- manipulate the host cell's response to other viruses. The UC Berkeley team has not yet photographed any huge phages, so all are depicted resembling the most common type of phage, T4. Credit: UC Berkeley image courtesy of Jill Banfield lab 据美国 加州大学 - 伯克利分校 ( University of California - Berkeley ) 2020 年 2 月 12 日提供的消息,该校研究人员研究发现,巨大的食细菌病毒弥合了生命与非生命之间的鸿沟 ( 图 1 所示 ) 。图 1 描绘了感染细菌细胞的巨大噬菌体(红色,左侧)和正常噬菌体。巨大的噬菌体将其 DNA 注入宿主细胞,其中 Cas 蛋白(通常仅在细菌和古细菌中发现的 CRISPR 免疫系统的一部分)操纵宿主细胞对其他病毒的反应。加州大学伯克利分校( UC Berkeley )的团队尚未拍摄任何巨大的噬菌体,因此所有照片均与最常见的噬菌体 T4 类似。 科学家发现了数百种异常大的,能杀死细菌的病毒,它们通常具有与生物体相关的功能,从而模糊了活微生物体( living microbes )与病毒机器( viral machines )之间的界线。 这些噬菌体 (phages) 是 bacteriophages (噬菌体)的缩写,所谓的噬菌体是因为它们 “ 吞噬 ” 细菌而得名。噬菌体具有一定的大小和复杂性,被认为是生命的典型特征,它们携带着细菌中通常发现的许多基因,并利用这些基因来对付它们的细菌宿主。 加州大学伯克利分校的研究人员及其合作者,通过搜寻庞大的 DNA 数据库来发现这些巨大的噬菌体。这些数据库的 DNA 是来自近 30 个不同的地球环境 , 从早产儿和孕妇的内脏到西藏温泉 (Tibetan hot spring) 、南非生物反应器( South African bioreactor )、医院病房、海洋、湖泊以及地下深处等。他们总共确定了 351 种不同的巨大噬菌体,它们的基因组比捕食单细胞细菌的病毒的平均基因组大四倍或更多倍。其中有迄今为止最大的噬菌体:其 基因组长 73.5 万个碱基对,比平均噬菌体大近 15 倍。这个最大的已知噬菌体基因组比许多细菌的基因组大得多。 加州大学伯克利分校地球与行星科学、环境科学、政策与管理教授吉莉安· 班菲尔德( Jillian F. Banfield )说: “ 我们正在探索地球的微生物群,有时会出现意想不到的事情。这些细菌病毒是生物学的一部分 , 也是复制实体( replicating entities )的一部分,对此我们鲜为人知。 ” 吉莉安 · 班菲尔德也是有关该发现论文的通讯作者,该论文于 2020 年 2 月 12 日在《 自然 》( Nature )杂志上发表—— Basem Al-Shayeb, Rohan Sachdeva, Lin-Xing Chen, Fred Ward, Patrick Munk, Audra Devoto, Cindy J. Castelle, Matthew R. Olm, Keith Bouma-Gregson, Yuki Amano, Christine He, Raphaël Méheust, Brandon Brooks, Alex Thomas, Adi Lavy, Paula Matheus-Carnevali, Christine Sun, Daniela S. A. Goltsman, Mikayla A. Borton, Allison Sharrar, Alexander L. Jaffe, Tara C. Nelson, Rose Kantor, Ray Keren, Katherine R. Lane, Ibrahim F. Farag, Shufei Lei, Kari Finstad, Ronald Amundson, Karthik Anantharaman, Jinglie Zhou, Alexander J. Probst, Mary E. Power, Susannah G. Tringe, Wen-Jun Li, Kelly Wrighton, Sue Harrison, Michael Morowitz, David A. Relman, Jennifer A. Doudna, Anne-Catherine Lehours, Lesley Warren, Jamie H. D. Cate, Joanne M. Santini, Jillian F. Banfield. Clades of huge phages from across Earth's ecosystems ( Open Access ). Nature , 2020. DOI: 10.1038/s41586-020-2007-4 , Published:12 February 2020 . https://www.nature.com/articles/s41586-020-2007-4.pdf 吉莉安 · 班菲尔德说: “ 一方面,这些巨大的噬菌体弥合了无生命的噬菌体与细菌和古细菌之间的鸿沟。似乎存在着成功的生存策略,我们认为这些策略就是传统病毒和传统活生物体之间的杂交体。 ” 具有讽刺意味的是,在这些巨大的噬菌体所环绕的 DNA 中,是细菌用来对抗病毒的 CRISPR 系统的一部分。一旦这些噬菌体将其 DNA 注入细菌中,病毒 CRISPR 系统就会增强宿主细菌的 CRISPR 系统,可能主要针对其他病毒。 CRISPR ( Clustered regularly interspaced short palindromic repeats )被称为规律成簇间隔短回文重复,实际上就是一种基因编辑器,是细菌用以保护自身对抗病毒的一个系统,也是一种对付攻击者的基因武器。 加州大学伯克利分校的研究生巴塞姆 · 阿尔 - 沙耶布( Basem Al-Shayeb )说: “ 这些噬菌体如何改变了我们认为是细菌或古细菌的系统,利用它们的竞争使其自身受益,促进这些病毒之间的殴斗,这实在令人着迷。 ” 巴塞姆 · 阿尔 - 沙耶布和他的同事 罗翰·萨谢德瓦(Rohan Sachdeva )是《自然》杂志论文的共同第一作者。 新的 Cas 蛋白 巨大的噬菌体之一也能制造出一种类似于 Cas9 的蛋白质,这种蛋白质是加州大学伯克利分校的詹妮弗·杜德纳 (Jennifer Doudna) 和她的欧洲同事艾曼纽埃尔·沙彭特 (Emmanuelle Charpentier) 为基因编辑而改造的革命性工具 CRISPR-Cas9 的一部分。研究团队将这种小蛋白质称为 Cas Φ , 因为希腊字母大写Φ或小写φ , 传统上被用来表示噬菌体。 罗翰·萨谢德瓦说: “ 在这些巨大的噬菌体中,寻找基因组工程 新工具 的潜力很大。 ”“ 我们发现的许多基因都是未知的,它们无推定功能 (putative function) ,可能是工业、医学或农业应用中新蛋白质的来源。 ” 除了为噬菌体和细菌之间的持续战争提供新的见解之外,新发现还对人类疾病产生影响。通常,病毒在细胞之间携带基因,包括赋予抗生素抗性的基因。而且由于噬菌体会在细菌和古生菌生活的任何地方(包括人类肠道微生物群)发生,因此它们可以将破坏性基因带入定居人类的细菌中。 吉莉安 · 班菲尔德说: “ 某些疾病是由噬菌体间接引起的,因为噬菌体在涉及发病机制和抗生素耐药性的基因周围移动。而且,基因组越大,围绕这些基因的移动能力就越大,并且能够将不良基因传递给人类微生物群中细菌的可能性就越高。 ” 吉莉安 · 班菲尔德也是创新基因组学研究所( Innovative Genomics Institute, IGI )微生物研究的负责人。 对地球生物群落的测序( Sequencing Earth's biomes ) 吉莉安 · 班菲尔德探索细菌的多样性已经坚持了超过 15 年, 10 多年以来,她一直不懈努力。她说,古细菌( Archaea )是地球上不同环境中细菌和噬菌体的迷人表亲( fascinating cousins )。她的方法是对样本中的所有 DNA 进行测序,然后将片段拼接在一起,形成草图基因组,或者在某些情况下,将从未见过的微生物的基因组完全整理好,这些工作她都做到了。在此过程中,她发现许多新微生物的基因组极小,似乎不足以维持独立生命。相反,它们似乎依赖其他细菌和古细菌生存。一年前,她报告说,在我们的肠道和口中发现了一些最大的噬菌体(一个叫做 Lak 的噬菌体),它们在那里捕食肠道和唾液微生物。 新的《自然》( Nature )杂志上发表的论文,是对吉莉安 · 班菲尔德积累的所有宏基因组序列( metagenomic sequences )中的巨大噬菌体进行了更彻底的搜索,再加上全球研究合作伙伴提供的新的宏基因组。宏基因组来自狒狒( baboons )、猪、阿拉斯加麋鹿( Alaskan moose )、土壤样本、海洋、河流、湖泊以及地下水,其中包括一直饮用砷污染水的孟加拉国人( Bangladeshis )。 该团队确定了 351 个噬菌体基因组,它们的长度超过 200 千碱基 (kilobases , kb) ,是平均噬菌体基因组长度 (50 kb) 的四倍。他们能够确定 175 个噬菌体基因组的确切长度,其他噬菌体的长度则可能大于 200 kb 。完整的基因组之一,长 735 kb ,现已成为已知的最大 噬菌体 基因组。 尽管这些巨大噬菌体中的大多数基因编码是未知蛋白质,但研究人员仍能够鉴定出编码对该机制至关重要的蛋白质——称为核蛋白体( ribosome )的基因,该核蛋白体将信使 RNA 转化为蛋白质。这类基因通常不在病毒( viruses )中出现,仅发现于细菌或古细菌中。 研究人员发现了许多用于转移 RNA 的基因,这些基因携带氨基酸到核糖体中并被整合到新蛋白质中。加载和调节 tRNA 的蛋白质的基因;开启翻译甚至是核糖体自身片段的蛋白质的基因。 罗翰·萨谢德瓦说: “ 通常,将生命与非生命区分开来的是拥有核糖体和进行翻译的能力;这是区分病毒和细菌,非生命与生命的主要特征之一。 ”“ 一些大型噬菌体具有很多这种翻译机制,因此它们使这条界线有些模糊。 ” 巨大的噬菌体可能使用这些基因来重定向核糖体,从而以细菌蛋白为代价,复制更多自身蛋白。一些巨大的噬菌体还具有其他的遗传密码,即编码特定氨基酸的核酸三联体,可能会混淆解码 RNA 的细菌核糖体。 此外,一些新发现的巨大噬菌体携带了在各种细菌 CRISPR 系统中发现的 Cas 蛋白变体的 基因 ,例如 Cas9 , Cas12 , CasX 和 CasY 家族。 CasØ 是 Cas12 家族的一种变体。一些巨大的噬菌体也具有 CRISPR 阵列,这是细菌基因组中存储病毒 DNA 片段的区域,以备将来参考,从而使 细菌 能够识别返回的噬菌体,并动员其 Cas 蛋白靶向并将其粉碎。 吉莉安 · 班菲尔德说: “ 高水平的结论是,具有大型基因组的噬菌体在地球的整个生态系统中都非常突出,它们并不是一个生态系统的独特之处。 ”“ 与具有大基因组的噬菌体相关,这意味着它们是具有悠久的大基因组历史的血统。拥有大基因组是一种成功的生存策略,而我们却对此知之甚少。 ” 更多信息请注意浏览原文或者相关报道。 Researchers discover new viral strategy to escape detection Abstract Bacteriophages typically have small genomes 1 and depend on their bacterial hosts for replication 2 . Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200kilobases (kb), including a genome of 735kb, which is—to our knowledge—the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR–Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR–Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR–Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth’s ecosystems.
抗生素在控制细菌性传染病上立下了汗马功劳,但它们同时也是催生“超级细菌”(superbug)的催化剂。所谓超级细菌,就是那些生长繁殖不受特定抗生素抑制的特殊细菌,这些细菌中要么含有某种抗生素抗性基因编码质粒,如甲氧西林抗性金黄色葡萄球菌(MRSA)的NDM-1以及 碳青霉烯抗性肠杆菌科细菌(CRE) , 要么携带某种抗生素抗性突变 基因,如喹诺啉(沙星类)抗性的出血性大肠杆菌SHV-18基因。 对于上述超级细菌,最有效的抑杀方式是避免使用同类抗生素,而换用其他抗生素。最近,美国麻省理工学院的科学家想出了一条妙计,他们不用任何抗生素,而是以细菌的天敌——噬菌体来绞杀超级细菌,这正如农业上利用白僵菌防治玉米螟那样。 细菌中普遍存在“成簇规律间隔的短回文重复”( CRISPR )编辑系统,可以用来抵御噬菌体的入侵和寄生,因而被形象地称为细菌的“免疫系统”。由于CRISPR与噬菌体DNA同源,因而可以互补配对,从而被细菌的 “手术刀 ”—— Cas9 蛋白识别并被切割, 结果使噬菌体不能感染细菌。 为了利用CRISPR-Cas9系统,研究人员设计了两种载运体,一种是携带CRISPR质粒的基因工程细菌,还有一个是可以感染细菌并注入 CRISPR的 噬菌体颗粒。当他们把两种运载体分别导入含NDM-1基因的超级细菌后,可以通过碱基互补找到NDM-1基因所在的未知,从而让细菌的Cas9切割NDM-1基因,结果发现该法杀菌率高达99%! 聪明的读者大概想到了,这个方法并不是让天然噬菌体来杀死细菌,而是利用细菌抑制噬菌体感染的原理让细菌“自杀”,只不过是利用基因工程细菌和噬菌体把CRISPR引入到部分菌体中,并传播至整个细菌群体。目前已在大蜡螟中完成了活体杀菌试验,下一步将在 小鼠中评价其杀菌效果。不过,这种技术何时用于人体还有待时日,可能的方式是通过益生菌引入肠道。 Battling superbugs: Two new technologies could enable novel strategies for combating drug-resistant bacteria Date: September 21, 2014 Source: Massachusetts Institute of Technology Summary: Two new technologies could enable novel strategies for combating drug-resistant bacteria, scientists report. Most antibiotics work by interfering with crucial functions such as cell division or protein synthesis. However, some bacteria have evolved to become virtually untreatable with existing drugs. In the new study, researchers target specific genes that allow bacteria to survive antibiotic treatment. The CRISPR genome-editing system presented the perfect strategy to go after those genes, they report. A scanning electron micrograph depicts numerous clumps of methicillin-resistant Staphylococcus aureus bacteria, commonly referred to by the acronym MRSA. Credit: Janice Haney Carr/Centers for Disease Control and Prevention In recent years, new strains of bacteria have emerged that resist even the most powerful antibiotics. Each year, these superbugs, including drug-resistant forms of tuberculosis and staphylococcus, infect more than 2 million people nationwide, and kill at least 23,000. Despite the urgent need for new treatments, scientists have discovered very few new classes of antibiotics in the past decade. MIT engineers have now turned a powerful new weapon on these superbugs. Using a gene-editing system that can disable any target gene, they have shown that they can selectively kill bacteria carrying harmful genes that confer antibiotic resistance or cause disease. Led by Timothy Lu, an associate professor of biological engineering and electrical engineering and computer science, the researchers described their findings in the Sept. 21 issue of Nature Biotechnology . Last month, Lu's lab reported a different approach to combating resistant bacteria by identifying combinations of genes that work together to make bacteria more susceptible to antibiotics. Lu hopes that both technologies will lead to new drugs to help fight the growing crisis posed by drug-resistant bacteria. This is a pretty crucial moment when there are fewer and fewer new antibiotics available, but more and more antibiotic resistance evolving, he says. We've been interested in finding new ways to combat antibiotic resistance, and these papers offer two different strategies for doing that. Cutting out resistance Most antibiotics work by interfering with crucial functions such as cell division or protein synthesis. However, some bacteria, including the formidable MRSA (methicillin-resistant Staphylococcus aureus ) and CRE (carbapenem-resistant Enterobacteriaceae) organisms, have evolved to become virtually untreatable with existing drugs. In the new Nature Biotechnology study, graduate students Robert Citorik and Mark Mimee worked with Lu to target specific genes that allow bacteria to survive antibiotic treatment. The CRISPR genome-editing system presented the perfect strategy to go after those genes. CRISPR, originally discovered by biologists studying the bacterial immune system, involves a set of proteins that bacteria use to defend themselves against bacteriophages (viruses that infect bacteria). One of these proteins, a DNA-cutting enzyme called Cas9, binds to short RNA guide strands that target specific sequences, telling Cas9 where to make its cuts. Lu and colleagues decided to turn bacteria's own weapons against them. They designed their RNA guide strands to target genes for antibiotic resistance, including the enzyme NDM-1, which allows bacteria to resist a broad range of beta-lactam antibiotics, including carbapenems. The genes encoding NDM-1 and other antibiotic resistance factors are usually carried on plasmids -- circular strands of DNA separate from the bacterial genome -- making it easier for them to spread through populations. When the researchers turned the CRISPR system against NDM-1, they were able to specifically kill more than 99 percent of NDM-1-carrying bacteria, while antibiotics to which the bacteria were resistant did not induce any significant killing. They also successfully targeted another antibiotic resistance gene encoding SHV-18, a mutation in the bacterial chromosome providing resistance to quinolone antibiotics, and a virulence factor in enterohemorrhagic E. coli . In addition, the researchers showed that the CRISPR system could be used to selectively remove specific bacteria from diverse bacterial communities based on their genetic signatures, thus opening up the potential for microbiome editing beyond antimicrobial applications. To get the CRISPR components into bacteria, the researchers created two delivery vehicles -- engineered bacteria that carry CRISPR genes on plasmids, and bacteriophage particles that bind to the bacteria and inject the genes. Both of these carriers successfully spread the CRISPR genes through the population of drug-resistant bacteria. Delivery of the CRISPR system into waxworm larvae infected with a harmful form of E. coli resulted in increased survival of the larvae. The researchers are now testing this approach in mice, and they envision that eventually the technology could be adapted to deliver the CRISPR components to treat infections or remove other unwanted bacteria in human patients. High-speed genetic screens Another tool Lu has developed to fight antibiotic resistance is a technology called CombiGEM. This system, described in the Proceedings of the National Academy of Sciences the week of Aug. 11, allows scientists to rapidly and systematically search for genetic combinations that sensitize bacteria to different antibiotics. To test the system, Lu and his graduate student, Allen Cheng, created a library of 34,000 pairs of bacterial genes. All of these genes code for transcription factors, which are proteins that control the expression of other genes. Each gene pair is contained on a single piece of DNA that also includes a six-base-pair barcode for each gene. These barcodes allow the researchers to rapidly identify the genes in each pair without having to sequence the entire strand of DNA. You can take advantage of really high-throughput sequencing technologies that allow you, in a single shot, to assess millions of genetic combinations simultaneously and pick out the ones that are successful, Lu says. The researchers then delivered the gene pairs into drug-resistant bacteria and treated them with different antibiotics. For each antibiotic, they identified gene combinations that enhanced the killing of target bacteria by 10,000- to 1,000,000-fold. The researchers are now investigating how these genes exert their effects. This platform allows you to discover the combinations that are really interesting, but it doesn't necessarily tell you why they work well, Lu says. This is a high-throughput technology for uncovering genetic combinations that look really interesting, and then you have to go downstream and figure out the mechanisms. Once scientists understand how these genes influence antibiotic resistance, they could try to design new drugs that mimic the effects, Lu says. It is also possible that the genes themselves could be used as a treatment, if researchers can find a safe and effective way to deliver them. CombiGEM also enables the generation of combinations of three or four genes in a more powerful way than previously existing methods. We're excited about the application of CombiGEM to probe complex multifactorial phenotypes, such as stem cell differentiation, cancer biology, and synthetic circuits, Lu says. Story Source: The above story is based on materials provided by Massachusetts Institute of Technology . The original article was written by Anne Trafton. Note: Materials may be edited for content and length. Journal Reference : Robert J Citorik, Mark Mimee, Timothy K Lu. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases . Nature Biotechnology , 2014; DOI: 10.1038/nbt.3011
新的想法,特别是在一个新的领域里的新想法,未必有人引用。倒是在成熟领域的改进可能当时影响的人更多。开博后写了这些篇博文,唯一的精选博文竟是非常技术的一篇。而且不知道为什么好像点击比其他更有创意,影响更大的博文还高,到现在居然有四千多。真的是博文领域比较大的原因还是因为博文精选导致的误打误撞比较多,也看不清楚。倒是让我觉得科学网不只是玩标题党,也有些关心技术的人。哪怕只有点击数的1%的人真正在用这个方法,也有四十多个实验室了。既然是个不错的宣传方法,就继续宣传。还是应该给博主一个自己把博文排序的功能。 今天再说说我们做的找泛素连接酶底物的两个方法吧。 总结以前的方法太费时间了,也有综述,估计看了这个题目还准备点开看正文的人也都知道了。还是直入正题吧。 第一个方法是把活的表达了蛋白在表面的噬菌体文库直接扔到泛素化反应的体系中去。这时泛素化体系中的连接酶特异性地泛素化了文库中的几个噬菌体表面的蛋白。然后我们再把表面有泛素化蛋白的噬菌体富集起来,感染细菌,制备噬菌体,把噬菌体所带的插入基因测序就知道被这个连接酶泛素化的蛋白是什么了。这个实验还是需要一点前提条件的。就是这个连接酶除了表面表达的蛋白外不能泛素化噬菌体的其他蛋白。这个可以在放入噬菌体文库的实验前,先放入没有表面呈现蛋白的空噬菌体试一试。背景足够低就可以了。还有一个条件是噬菌体能够在泛素化的反应体系中保持活力和繁殖力。泛素化的体系不是特别难适应,噬菌体看样子没有问题能熬过那一段37度的时光,估计绝大部分噬菌体在表面蛋白又加上泛素后都还保持着繁殖力。整个策略如果加上一步负选择可以提前去掉文库中能够产生假阳性的克隆,后续验证效率更高。这个方法优点是比较简单快速高通量。整个过程见示意图。文章的链接在这里: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0076622#pone-0076622-g004 Screening E3 substrates using a live phage display library.pdf 第二个方法是如果连接酶有其他的蛋白结合结构域,可以先通过这结构域找连接酶的结合蛋白,然后再试试这个结合蛋白是不是底物。当然如果只结合而不是底物,那可能就是一个站着茅坑不拉屎的泛素化调解物,它拦住了其他底物被泛素的路。泛素化的调解物用其他的方法好像还很难找到。我没印象哪个方法可以高通量筛选泛素化的调节蛋白,如果查证真没有,其实以后可以专门开发一个。我们试的是一个有PDZ结构域的连接酶。先通过PDZ的结合特性找到可能结合的蛋白,然后表达出来试试是不是底物。因为丰度不高的底物不是和其他丰度高的底物在一起筛,这个方法可以找到量比较少的底物。验证起来一个一个克隆表达比较麻烦。我们就做了一个统一的可泛素化的Degron连上我们筛出来的识别位点做底物,初步做体外验证。这样效率高一些。进一步的验证实验再用整蛋白做,那时是底物的可能性已经增大了,复杂的实验可能也值得做了。 http://pubs.acs.org/doi/abs/10.1021/pr300674c A Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-Containing E3 .pdf 欢迎大家批评指正多提宝贵意见! 2014-5-4应读者要求贴上原文。