Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs

Background

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptive defence system that provides resistance against alien replicons such as viruses and plasmids. Spacers in a CRISPR cassette confer immunity against viruses and plasmids containing regions complementary to the spacers and hence they retain a footprint of interactions between prokaryotes and their viruses in individual strains and ecosystems. The human gut is a rich habitat populated by numerous microorganisms, but a large fraction of these are unculturable and little is known about them in general and their CRISPR systems in particular.

Results

We used human gut metagenomic data from three open projects in order to characterize the composition and dynamics of CRISPR cassettes in the human-associated microbiota. Applying available CRISPR-identification algorithms and a previously designed filtering procedure to the assembled human gut metagenomic contigs, we found 388 CRISPR cassettes, 373 of which had repeats not observed previously in complete genomes or other datasets. Only 171 of 3,545 identified spacers were coupled with protospacers from the human gut metagenomic contigs. The number of matches to GenBank sequences was negligible, providing protospacers for 26 spacers.Reconstruction of CRISPR cassettes allowed us to track the dynamics of spacer content. In agreement with other published observations we show that spacers shared by different cassettes (and hence likely older ones) tend to the trailer ends, whereas spacers with matches in the metagenomes are distributed unevenly across cassettes, demonstrating a preference to form clusters closer to the active end of a CRISPR cassette, adjacent to the leader, and hence suggesting dynamical interactions between prokaryotes and viruses in the human gut. Remarkably, spacers match protospacers in the metagenome of the same individual with frequency comparable to a random control, but may match protospacers from metagenomes of other individuals.

Conclusions

The analysis of assembled contigs is complementary to the approach based on the analysis of original reads and hence provides additional data about composition and evolution of CRISPR cassettes, revealing the dynamics of CRISPR-phage interactions in metagenomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-202) contains supplementary material, which is available to authorized users.     

人类口腔微生物组学研究:现状、挑战及机遇

全球超过一半的人口患有口腔疾病,其医护费用与全球十大常见死亡病因的花费相当,而且口腔感染与早产、动脉粥样硬化、肝硬化、糖尿病、阿尔茨海默病等全身性或慢性疾病显著相关,因此,口腔微生物组一直是人类微生物组计划的主要研究对象之一。与人体其他部位比较,口腔微生物组研究具有取样快捷、宿主反应表征方便、干预手段直接有效等特点;同时,超过65%的口腔细菌类群已可培养,诸多代表性菌株的全基因组信息已破译。因此口腔微生物组在菌群内部调控网络及其与宿主互作机制、局部感染对远隔器官的影响机制、以及基于菌群的慢病早期预警等微生物组研究核心科学问题上具备作为模式研究体系与技术示范对象的重要优势。本文在分析口腔微生物组学国际、国内研究现状的基础上,建议尽快启动中国人口腔微生物组计划(China human oral microbiome project,CHOMP),通过产学研协同攻关,开拓基于口腔菌群的口腔及全身系统性疾病的个体化预防、诊断及治疗策略。     

Association between living environment and human oral viral ecology

The human oral cavity has an indigenous microbiota known to include a robust community of viruses. Very little is known about how oral viruses are spread throughout the environment or to which viruses individuals are exposed. We sought to determine whether shared living environment is associated with the composition of human oral viral communities by examining the saliva of 21 human subjects; 11 subjects from different households and 10 unrelated subjects comprising 4 separate households. Although there were many viral homologues shared among all subjects studied, there were significant patterns of shared homologues in three of the four households that suggest shared living environment affects viral community composition. We also examined CRISPR (clustered regularly interspaced short palindromic repeat) loci, which are involved in acquired bacterial and archaeal resistance against invading viruses by acquiring short viral sequences. We analyzed 2 065 246 CRISPR spacers from 5 separate repeat motifs found in oral bacterial species of Gemella, Veillonella, Leptotrichia and Streptococcus to determine whether individuals from shared living environments may have been exposed to similar viruses. A significant proportion of CRISPR spacers were shared within subjects from the same households, suggesting either shared ancestry of their oral microbiota or similar viral exposures. Many CRISPR spacers matched virome sequences from different subjects, but no pattern specific to any household was found. Our data on viromes and CRISPR content indicate that shared living environment may have a significant role in determining the ecology of human oral viruses.     

口腔微生态失衡导致口腔异味的研究

目的介绍口腔微生态失调与口腔疾病的关系,口腔疾病出现口腔气味异常的机制。综合分析口腔微生态与口腔健康的研究动态及意义。展望口腔微生态调整在口腔疾病治疗中的作用研究,尤其在消除口腔异昧的积极作用。方法由第一、二作者应用计算机通过pubmed检索NCBI数据库1992年至2007年相关文献,检索词为“Micro ecology”,限定语言种类为“English”;同时检索CNKI全文数据库、维普全文数据库1995年至2007年相关文献,检索词为“微生态”,限定语言种类为中文。纳入标准：文章内容与口腔微生态相关的研究、以及在口腔疾病研究领域有关。排除标准：较陈旧的文献和重复研究。结果共收集到106篇相关文献,21篇文献纳入本文,其中,19篇为综述和述评类文献,19篇为中文杂志,2篇为外文杂志。结论致病菌大量生长繁殖,口腔微生态失调,菌斑内微生物之间以及机体与菌斑之间相互作用分解蛋白产生硫化物,这些代谢产物包括H2S、CH3SH、CH3SCH3、吲哚、甲基吲哚、挥发脂肪酸和聚胺等发出刺激性气味,产生口腔异味。治疗口腔异味应该考虑平衡口腔微生态,调整口腔菌群。口腔异味的治疗又有利于口腔菌群平衡。提示我们治疗口腔疾病应该考虑口腔微生态平衡。     

Growth, incidence and activities of dissimilatory sulfate-reducing bacteria in the human oral cavity

Abstract Viable counts and activities of sulfate-reducing bacteria were determined in the oral cavities of 12 healthy volunteers. Of these, 10 harboured viable sulfate-reducing bacteria populations. Six separate sites were sampled: the posterior tongue, anterior tongue, mid buccal mucosa, vestibular mucosa, supragingival plaque and subgingival plaque. Sulfate-reducing bacteria occurred in all areas, with the highest incidence in supragingival plaque. Viable counts and sulfate-reducing activities in each of the regions varied from 0 to 10⁸ cfu (g wet weight)⁻¹ and from 0 to 50 nmol (g wet weight) ⁻¹ h⁻¹, respectively. As sulfate-reducing bacteria can be detected in the oral cavity, they may potentially be involved in terminal oxidative processes carried out by the microflora of the mouth.     

CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*

病毒感染相关疾病严重威胁人类的健康,目前的抗病毒疗法难以治愈慢性病毒性感染引起的一些疾病,如获得性免疫缺陷综合征和乙型肝炎等,因此迫切需要新的治疗方法。可直接靶向遗传物质的基因编辑技术或将成为对抗病毒的有力工具。作为一种可编程的核酸酶介导的新型基因编辑技术,CRISPR/Cas9系统因其具有编辑效率高、操作简单、成本低、适用范围广等优点,而成功应用于多种人类相关疾病的研究中,也为病毒感染相关疾病的研究以及开发新的治疗方法提供了新的技术手段。主要对CRISPR/Cas9系统的作用机制以及在人类常见的病毒感染相关疾病治疗研究中的最新进展进行综述。     

醋酸菌中CRISPR位点的比较基因组学与进化分析

CRISPR (Clustered regularly interspaced short palindromic repeats)是近几年发现的一种广泛存在于细菌和古菌中,能够应对外源DNA干扰(噬菌体、病毒、质粒等),并提供免疫机制的重复序列结构。CRISPR系统通常由同向重复序列、前导序列、间隔序列和CRISPR相关蛋白组成。本研究以醋酸发酵中常见3个属醋杆菌属(Acetobacter)、葡糖醋杆菌属(Gluconacetobacter)和葡糖杆菌属(Gluconobacter)的48个菌株为研究对象,通过其基因组上CRISPR相关基因序列的生物信息学分析,探索CRISPR位点在醋酸菌中的多态性及其进化模式。结果表明48株醋酸菌中有32株存在CRISPR结构,大部分CRISPR-Cas结构属于type I-E和type I-C类型。除了葡糖杆菌属外,葡糖醋杆菌属和醋杆菌属中的部分菌株含有II类的CRISPR-Cas系统结构(CRISPR-Cas9)。来自不同属菌株的CRISPR结构中重复序列具有较强的保守性,而且部分菌株CRISPR结构中的前导序列具有保守的motif (与基因的转录调控有关)及启动子序列。进化树分析表明cas1适合用于醋酸菌株的分类,而不同菌株间cas1基因的进化与重复序列的保守性相关,预示它们可能受相似的功能选择压力。此外,间隔序列的数量与噬菌体数量及插入序列(Insertion sequence, IS)数量有正相关的趋势,说明醋酸菌在进化过程中可能正不断受新的外源DNA入侵。醋酸菌中CRISPR结构位点的分析,为进一步研究不同醋酸菌株对醋酸胁迫耐受性差异及其基因组稳定性的分子机制奠定了基础。     

CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*

病毒感染相关疾病严重威胁人类的健康,目前的抗病毒疗法难以治愈慢性病毒性感染引起的一些疾病,如获得性免疫缺陷综合征和乙型肝炎等,因此迫切需要新的治疗方法。可直接靶向遗传物质的基因编辑技术或将成为对抗病毒的有力工具。作为一种可编程的核酸酶介导的新型基因编辑技术,CRISPR/Cas9系统因其具有编辑效率高、操作简单、成本低、适用范围广等优点,而成功应用于多种人类相关疾病的研究中,也为病毒感染相关疾病的研究以及开发新的治疗方法提供了新的技术手段。主要对CRISPR/Cas9系统的作用机制以及在人类常见的病毒感染相关疾病治疗研究中的最新进展进行综述。     

沙门氏菌CRISPR位点的结构特征比较

成簇规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR),是存在于多数细菌和古菌中的遗传结构,能够有效防御外源DNA的入侵(质粒、噬菌体等),进而防御外源基因的水平转移。【目的】本研究以沙门氏菌属中常见的鸡伤寒沙门氏菌(Salmonella gallinarum)、鼠伤寒沙门氏菌(Salmonella typhimurium)、猪霍乱沙门氏菌(Salmonella choleraesuis)以及肠炎沙门氏菌(salmonella enteritidis)等30个菌株为研究对象。探索CRISPR位点在不同沙门氏菌种中的结构差异。【方法】通过生物信息学的方法比较间隔序列与插入序列的同源性以及CRISPR位点与质粒数量关系。【结果】30株沙门氏菌中均存在CRISPR结构,包括CRISPR位点61个以及可疑位点12个。重复序列和cas1基因均不能作为这4类细菌的分类依据。【结论】虽然我们发现CRISPR位点数量与间隔区数量和质粒数量之间均不存在统计学关系,但间隔序列整合子、耐药基因等移动遗传原件具有一定的同源性,说明沙门氏菌在进化过程中不断受外源基因的侵袭。     

Comparative analysis of CRISPR loci in different Listeria monocytogenes lineages

Listeria monocytogenes, an important food-borne pathogen, causes high mortality rate of listeriosis. Pan-genomic comparisons revealed the species genome of L. monocytogenes is highly stable but not completely clonal. The population structure of this species displays at least four evolutionary lineages (I–IV). Isolates of different lineages displayed distinct genetic, phenotypic and ecologic characteristics, which appear to affect their ability to be transmitted through foods and to cause human disease, as well as their ability to thrive in markedly phage-rich environments. CRISPR (clustered regularly interspaced short palindrome repeats), a recently described adaptive immunity system, not only confers defense against invading elements derived from bacteriophages or plasmids in many bacteria and archaeal, but also displays strains-level variations in almost any given endowed species. This work was aimed to investigate CRISPR diversity in L. monocytogenes strains of different lineages and estimated the potential practicability of the CRISPR-based approach to resolve this species’ biodiversity. Only a third of strains contained all three CRISPR loci (here defined as LMa, LMb and LMc) at same time. Combined the strain-level variations in presence/absence of each CRISPR locus and its relative size and spacer arrangements, a total of 29 CRISPR genotypes and 11 groups were defined within a collection of 128 strains covering all serotypes. The CRISPR-based approach showed powerful ability to subtype the more commonly food-borne isolates of serotype 1/2a (lineage II) and serotypes 1/2b (lineage I), but limited by the absence of typical CRISPR structure in many lineage I isolates. Strikingly, we found a long associated cas1 gene as well as two self-targeting LMb spacers accidently homologous with endogenous genes in a fraction of serotype 1/2a isolations, demonstrated that CRISPR I B system might involve in bacterial physiology besides antiviral immunity.     

Isolation and identification of the oral bacteria and their characterization for bacteriocin production in the oral cavity

Oral cavity is a diverse ecosystem which harbors immense diversity of microorganisms like fungi, virus and bacteria. Some of these microorganisms are involved in causing multiple infections. Oral flora is continuously changing due to connection with the external environment and produce bacteriocin against each other to compete for nutrient in this mini ecosystem. Current study was aimed to explore and compare the bacterial fauna of both healthy and non-healthy dental samples, by isolation and identification with biochemical tests to characterize the bacteriocin production. During study 120 swabs were taken from both healthy and unhealthy subjects. Samples were collected from the dental clinics of Makkah City, in sterile eppendorfs containing 1 ml nutrient broth, and were incubated overnight using shaking incubator. Bacteria were isolated following identification through Gram staining, microscopy and biochemical test. Total 15 strains of bacteria were isolated during the study amongst which 8 strains were gram positive and 7 strains were gram negative. The most dominant species of the gram positive strains was Streptococcus pneumoniae (n = 26). On the other hand, Escherichia coli (n = 26) was the prominent specie amongst the gram negative strains. Overall, the dominated family was Enterobacteriaceae (19.36%) followed by Streptococcaceae with 13.83% abundance. One of the most cariogenic strain Klebsiella pneumoniae (n = 14) was also isolated. The bacterial strain diversity between these two type of ecosystem was approximately the same, with slight variation in Shannon (HS:2.627187, NHS:2.653594) and Simpson diversity (HS:0.923461, NHS: 0.92684) index. The current research revealed that bacteriocin production in the Enterobacter species was prominent against Escherichia coli and Klebsiella pneumoniae. Apart from this other strains like Klebsiella pneumoniae and Exiguobacterium spp were also able to produce bacteriocin against Enterobacter species and Bacillus cereus respectively.     

Beyond microbial community composition: functional activities of the oral microbiome in health and disease

The oral microbiome plays a relevant role in the health status of the host and is a key element in a variety of oral and non-oral diseases. Despite advances in our knowledge of changes in microbial composition associated with different health conditions the functional aspects of the oral microbiome that lead to dysbiosis remain for the most part unknown. In this review, we discuss the progress made towards understanding the functional role of the oral microbiome in health and disease and how novel technologies are expanding our knowledge on this subject.     

Characteristics of human oral microbiome and its non-invasive diagnostic value in chronic kidney disease

Background: Morbidity of chronic kidney disease (CKD) is increased, with many complications and high mortality rates. The characteristics of oral microbiome in CKD patients have not been reported. This study aims to analyze the oral microbiome, and to demonstrate the potential of microbiome as noninvasive biomarkers for CKD patients.Methods: The study collected 253 oral samples from different regions of China (Central China and East China) prospectively and finally 235 samples completed Miseq sequencing, including 103 samples from CKD patients and 132 healthy controls (HCs).Results: Compared with HCs (n=88), the oral microbial diversity in CKD patients (n=44) was increased. Fourteen genera including Streptococcus, Actinomyces and Leptotrichia were enriched, while six genera including Prevotella and Haemophilus were decreased in CKD patients. Moreover, 49 predicted microbial gene functions including arginine metabolism and tryptophan metabolism increased, while 55 functions including Ribosome and DNA repair recombination proteins decreased. Furthermore, correlation analysis demonstrated that 38 operational taxonomic units (OTUs) were closely related to 5 clinical indicators of CKD. Notably, 7 optimal biomarkers were identified using random forest model, and the classifier model respectively reached an area under the curve (AUC) of 0.9917 and 0.8026 in the discovery and validation phase, achieving a cross-region validation.Conclusions: We first illustrated the characteristics of the oral microbiome of patients with CKD, identified the potential of oral microbial makers as noninvasive tools for the diagnosis of CKD and achieved cross-region validation.     

The fluid mechanics of bolus ejection from the oral cavity

The squeezing action of the tongue against the palate provides driving forces to propel swallowed material out of the mouth and through the pharynx. Transport in respose to these driving forces, however, is dependent on the material properties of the swallowed bolus. Given the complex geometry of the oral cavity and the unsteady nature of this process, the mechanics governing the oral phase of swallowing are not well understood. In the current work, the squeezing flow between two approaching parallel plates is used as a simplified mathematical model to study the fluid mechanics of bolus ejection from the oral cavity. Driving forces generated by the contraction of intrinsic and extrinsic lingual muscles are modeled as a spatially uniform pressure applied to the tongue. Approximating the tongue as a rigid body, the motion of tongue and fluid are then computed simultaneously as a function of time. Bolus ejection is parameterized by the time taken to clear half the bolus from the oral cavity, t_1/2. We find that t_1/2 increases with increased viscosity and density and decreases with increased applied pressure. In addition, for low viscosity boluses (μapproximately 1000 cP), viscosity dominates. A transition region between these two regimes is found in which both properties affect the solution characteristics. The relationship of these results to the assessment and treatment of swallowing disorders is discussed.     

基于CRISPR/dCas9技术活细胞成像基因组位点的研究进展

基因组的三维空间组织在调节其功能、维持表观遗传和稳定性方面起着重要作用.虽然基于荧光原位杂交的标记技术或单细胞显微观察等方法对此进行了广泛研究,但是细胞内部的时空动态在很大程度上仍难以捉摸.目前标记和观察活细胞中特定内源基因组位点的方法操作难度大且对生物的侵入性伤害较大.CRISPR的发现彻底改变了基因组工程领域.除了...     

Pigmented Prevotella species in the periodontally healthy oral cavity

Abstract Pigmented Prevotella spp. have been connected with oral infections as well as being part of the healthy gingival flora. The aim of this study was to determine the presence of pigmented Prevotella spp. in saliva and gingival crevice samples from periodontally healthy adults. Twelve Caucasian female subjects (mean age 28 years, range 21–36 years) with no pockets ≥ 4 mm, nor bleeding after probing were selected for this study. Paraffin-stimulate saliva was collected first; then, a pooled subgingival bacterial sample was taken with a sterile curette from mesiobuccal surfaces of all first molars. The samples were inoculated on to non-selective and selective media and incubated anaerobically. The most frequent species isolated were Pr. melaninogenica, Pr. intermedia and Pr. loescheii , found in 11, ten and nine subjects, respectively. The mean percentages of the total cultivable anaerobic microflora in salivary/subgingival samples were 14.7/0.6 for Pr. melaninogenica , 3.1/5.3 for Pr. intermedia and 2.6/1.2 for Pr. loescheii. Pr. denticola was found in one saliva sample and Pr. corporis , in two subgingival samples only. The number of different pigmented Prevotella spp. in the same mouth was 2–4 (mean 2.75). In conclusion, Pr. melaninogenica, Pr. intermedia and Pr. loescheii seem to be common microorganisms in the periodontally healthy oral cavity.