人体肠道噬菌体的研究与应用

肠道是人体内微生物定殖最丰富的部位。近年来,随着肠道菌群与人体健康疾病关联研究的蓬勃发展,肠道噬菌体也逐渐引起关注。然而,相关信息技术和实验技术发展的滞后在一定程度上限制了肠道噬菌体的科学研究进程。因此,本文首先回顾了近几年来肠道噬菌体研究领域所开发或采用的计算和实验方法,包括噬菌体的测序数据分析和噬菌体的分离纯化等。随后,本文就肠道噬菌体的分类、肠道内噬菌体与细菌的互作及肠道噬菌体在人体疾病干预中的应用展开了讨论。最后,本文展望了肠道噬菌体研究在数据和实体资源、信息和实验技术、与肠道菌群的互作、干预和治疗人体疾病各方面的一系列挑战和机遇。     

微生物与生命健康专题序言

人体微生物组是指人体内由微生物组成的共生生态群落,其动态平衡与人体健康密切相关。微生物组被公认为可在人体中起调节免疫、代谢、消化吸收作用的重要“器官”,可与包括肺、肠道、阴道、大脑在内的多个器官产生联系,并逐步成为治疗癌症、冠心病、神经系统疾病等疑难杂症的潜在靶点。近年来,随着微生物组测序与分析技术的飞速发展,从微生物组角度发现人体微生物与多种疾病的关系并探索新的治疗方法已成为国际科研的热点与前沿。为了进一步促进人体微生物在生命健康领域的研究,《生物工程学报》特组织出版专题,重点阐述了人体微生物组的研究方法、人体微生物组与疾病以及干预方法等方面的研究进展,为推动微生物组在生命健康领域的快速发展提供理论基础。     

人体肠道病毒组的研究进展

人体肠道微生物包括细菌、真菌、古细菌和病毒等，肠道病毒组的基因组约占肠道微生物总DNA的5.8%，病毒的数量在所有肠道微生物中排名第二，且多样性高。受高比例细菌基因组的影响，很难直接从宏基因组数据中深入分析肠道病毒组。随着病毒样颗粒（VLPs）富集技术的出现以及高通量测序技术的迅速发展，近年来对肠道病毒组的研究越来越多。基于VLPs，发现真核病毒、植物相关病毒和噬菌体是肠道病毒组的主要组成部分，而且未知病毒序列占比高达81%～93%。现阶段病毒组富集扩增方法主要有超速离心、单引物扩增和多重置换扩增等。疾病特异性研究表明，炎症性肠病、酒精性肝病和2型糖尿病等与饮食及肠道病毒组改变相关。本综述回顾了近十年对人类肠道病毒组的研究，总结归纳了目前肠道病毒组的构成、研究方法及与人体健康的相关性，探讨了未来肠道病毒组的研究方向，力求为后续的研究提供新思路。     

微生物组学研究的“淘金时代”

人体及动物肠道中生存着数量庞大的共生微生物;这些微生物无时无刻不参与着宿主的生命活动。揭示这些共生微生物在宿主体内的变化规律、与宿主之间的依存和博弈关系等,将使人类更加全面的认知高等生物体的生命本质。本专刊从肠道微生物与疾病、肠道微生物群落结构、肠道微生物与宿主互作、肠道微生物资源和肠道微生物研究方法 5个层面展示了我国科研工作者在肠道微生物研究领域的新进展及新观点。     

口腔微生物与全身健康研究进展

口腔微生物是定植于人体口腔的微生物集合。众多研究证实,口腔微生物与多种口腔感染性疾病及系统性疾病紧密相关。随着“人类微生物组计划”及其他微生物宏基因组学相关项目的开展,人们对口腔微生物群落的认识不断深入。本文基于最新研究进展,就口腔微生物的组成、演替特点、与口腔和全身系统性疾病的关系及与肠道微生物的交互作用进行综述。     

人体肠道微生物多样性和功能研究进展

人体肠道中庞大而复杂的微生物群落对人体自身代谢表型有深远的影响.肠道微生物群落在亚种或菌株水平上表现出极大的多样性.利用微生物分子生态学、元基因组学和代谢组学研究方法,发现肠道微生物与宿主表现出共进化的特点,肠道微生物群落及其基因组为宿主提供了互补的遗传和代谢功能,表现出互惠共生关系.但是,肠道微生物群落中影响宿主代谢表型的关键功能菌鉴定及其作用模式问题仍然悬而未决,综合运用多种高通量研究方法和多维数据分析方法可能成为解决这个问题的突破口.     

肠道微生物蛋白糖基化修饰的研究进展

肠道微生物在维持人体健康和诱导疾病的发展中扮演着重要角色,其蛋白糖基化修饰深刻影响着宿主的各项生命活动。本文从糖组学的角度出发,讨论并分析了肠道微生物的组成、作用,以及肠道微生物群中代表性细菌的糖基化模式及其密切相关的生理功能,发现及归纳了糖基化对肠道微生物功能和活动的调节方式,为相关疾病的研究及诊治提供了一个新的思路。     

浅议中医体质与肠道微生态的关系及个体化医疗

中医体质是人体生命过程中先天禀赋和后天获得基础上所形成的形态结构,具有生理功能和心理状态方面综合的、相对稳定的固有特质。中医体质分型更好地阐述了独立个体疾病的易患因素和疾病演变的倾向性,从而更好地诠释中医体系的个体化医疗。人体肠道微生物总量达100万亿,占人体总微生物量的78.67%,肠道微生态系统是人体最重要的微生态系统。肠道微生态系统可以更好地掌握和利用精准遗传学信息来防病治病、促进健康,从而更好地诠释西医体系的个体化医疗。基于中医体质理论与肠道微生态学相结合的个体化医疗兼顾中西医学理论基础及治疗方案,其关联性的研究使现代科学理论方法和技术在中医学研究中有了用武之地。     

采用RAPD-PCR技术评估果蝇肠道噬菌体多样性

噬菌体作为微生物群落结构和功能的主要驱动力之一,研究其在动物肠道中的生态进化过程具有重要意义。本研究以黑腹果蝇为模型,探究以随机多态性DNA聚合酶链式反应(RAPD-PCR)病毒指纹技术评估肠道噬菌体的多样性。结果表明,野生型黑腹果蝇肠道噬菌体的多样性高于InR突变体黑腹果蝇。InR突变体果蝇在孵化为成虫后,可能经历一段短期的肠道噬菌体扰动,与第3天相比,第15天的肠道噬菌体多样性显著降低,而健康的野生型果蝇则没有明显变化。进一步利用落射荧光显微镜测量噬菌体和细菌的丰度,在第3天和15天,野生型果蝇游离噬菌体和细菌均处于稳定状态。第3天InR突变体果蝇肠道细菌的丰度显著降低,病毒与细菌丰度的比值(VBR)明显高于其他组;第15天InR突变型果蝇噬菌体的多样性降低,VBR明显低于各处理组。本文证实了InR突变体果蝇肠道噬菌体生态学特征与野生型存在一定的差异,也表明RAPD-PCR病毒指纹技术可以实现对肠道病毒多样性的快速评估。     

医学遗传学2.0:导致人类慢病的主因可能首先是人体共生微生物基因异常,其次才是人类基因异常

当前慢病高发的现实对"健康中国2030"战略目标的实现提出了巨大挑战。虽然众多医疗机构和政府管理部门付出巨大努力,然而如果仍然沿袭现有慢病防控模式和医疗改革理念,恐怕很难在近期内实现慢病防控的突破,迫切需要引入新思路,才有可能破解慢病高发这个难题。根据近年来国内外大量报道人体共生微生物尤其是肠道菌群与人体多种慢病之间存在密切相关性甚至因果性的研究进展,以及在此启发下我们实验室通过大量研究发现"饥饿源于菌群",结合诸多文献报道证明通过调控肠道菌群微生态可改善多种慢病,为"慢病源于菌群"提供了重要依据,从而提出"医学遗传学2.0"(Medical genetics 2.0, MG2.0)的概念,其核心思想是将复杂性疾病(主要指慢病)的致病因素优先归因于人体共生微生物尤其是肠道菌群基因组异常,而人类基因组异常则是跟随前者发生顺应性改变的结果,即人体共生微生物基因组异常是慢病的主要矛盾,人类自身的基因组异常是慢病的次要矛盾,两套基因组通过联立交互作用,最终导致人体慢病持续发展。如果只是通过纠正人类基因组异常,而忽视了纠正菌群基因组异常,则难以从根本上治疗慢病,因为异常的菌群基因组仍然会持续影响人体健康。因此,在慢病防控方面,建议医学遗传学领域的研究重点可向肠道菌群等人体共生微生物领域进行深化,广泛开展以人体共生微生物尤其是肠道菌群基因组为主、人类基因组为辅的人菌双基因组关联分析研究,建立不同慢病的菌群图谱(含基因组学、转录组学、蛋白质组学、代谢组学以及生命组学等相关研究),并研究纠正异常菌群图谱的方法(含靶向肠道菌群的新药研发),为慢病防控找到新出路。     

肠道噬菌体组生物信息学分析方法的研究进展

噬菌体是感染细菌的病毒,广泛存在于各类环境中。由于传统实验研究的局限性及噬菌体基因的特异性,导致对肠道噬菌体的研究很少。随着宏基因组测序技术的发展和各种生物信息分析软件的开发,可以通过噬菌体组学,加深对肠道噬菌体的认识。噬菌体组分析流程主要包括原始数据质量控制和预处理,病毒基因组序列的拼接组装,类病毒颗粒的筛选和系统分类注释以及进化分析和预测相应宿主细菌。本文对噬菌体组分析流程和其中所需要的常用生物信息分析工具和数据库进行详细的介绍,可以为肠道噬菌体研究以及相关的研究人员提供参考。     

Bacteriophages and genetic mobilization in sewage and faecally polluted environments

Bacteriophages are one of the most abundant entities on the planet and are present in high concentrations within humans and animals, mostly in the gut. Phages that infect intestinal bacteria are released by defecation and remain free in extra‐intestinal environments, where they usually persist for longer than their bacterial hosts. Recent studies indicate that a large amount of the genetic information in bacterial genomes and in natural environments is of phage origin. In addition, metagenomic analysis reveals that a substantial number of bacterial genes are present in viral DNA in different environments. These facts support the belief that phages can play a significant role in horizontal gene transfer between bacteria. Bacteriophages are known to transfer genes by generalized and specialized transduction and indeed there are some examples of phages found in the environment carrying and transducing genes of bacterial origin. A successful transduction in the environment requires certain conditions, e.g. phage and bacterial numbers need to exceed certain threshold concentrations, the bacteria need to exist in an infection‐competent physiological state, and lastly, the physical conditions in the environment (pH, temperature, etc. of the supporting matrix) have to be suitable for phage infection. All three factors are reviewed here, and the available information suggests: (i) that the number of intestinal bacteria and phages in faecally contaminated environments guarantees bacteria–phage encounters, (ii) that transduction to intestinal bacteria in the environment is probable, and (iii) that transduction is more frequent than previously thought. Therefore, we suggest that phage‐mediated horizontal transfer between intestinal bacteria, or between intestinal and autochthonous bacteria in extra‐intestinal environments, might take place and that its relevance for the emergence of new bacterial strains and potential pathogens should not be ignored.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.     

Results of typing staphylococci isolated from cows and their milk products using the basic set of phages and local phages]

The basic set of phages recommended for typing staphylococci from cattle and also of local phages were approbated. Staphylococci cultures (950 in all) isolated in various regions of the Soviet Union from milk, milk produce and from cows suffering from mastitis were studied. Percentage of cultures typed by the phages of the basic set proved to be 78.3. Thirty different phage patterns were revealed among staphylococcal cultures lysed by phages. Lytic activity was the greatest in phages of group IV of the basic set. It is suggested that local phage 34k can be used as an additional phage permitting to subdivide the prevailing phage types within group IV into a number of new ones.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of the ϕKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ?KZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ?KZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an “inner body” was detected, which points to specific DNA package intrinsic to phages of ?KZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ?KZ-like phages in respect to detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa ?KZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination (“gene shuffling”) between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the ?KZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ?KZ composition. The results of the study are discussed in terms of the evolution of these phages.     

Synonymous codon usage in forty staphylococcal phages identifies the factors controlling codon usage variation and the phages suitable for phage therapy

The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2 may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be employed in curing staphylococcal infections.     

Search for destruction factors of bacterial biofilms: Comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products

Comparison of Pseudomonas putida group of phages attributed to five species (af, ?15, ?27, ?2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.     

Bacteriophage translocation

The occurrence of phages in the human body, especially in the gastrointestinal tract, raises the question of their potential role in the physiology and pathology of this system. Especially important is the issue of whether phages can pass the intestinal wall and migrate to lymph, peripheral blood, and internal organs and, if so, the effects such a phenomenon could have (such passage by bacteria, known as bacterial translocation, has been shown to cause various disturbances in humans, from immune defects to sepsis). Available data from the literature support the assumption that phage translocation can take place and may have some immunomodulatory effects. In addition, phages of the gut may play a protective role by inhibiting local immune reactions to antigens derived from gut flora.     

Defective bacteriophages

Naturally occurring defective phage particles, which do not form plaques on any known host, but have a restricted host killing range, appear to be widely distributed. The defective phages are produced spontaneously but can be induced, at much higher levels, by chemical and physical agents which interfere with metabolism or structure of DNA. The defective phages discussed in this article have been divided into various categories on the basis of their structural complexity, which ranges from what appears to be phage tail components through to intact phage particles, and the source of the DNA packaged into the heads of the phage-like particles. The evolution of the defective phages is discussed and the possibility is entertained that they may have originated from temperate phages.