RESUMO
The human oral microbiota is highly diverse and has a complex ecology, comprising bacteria, microeukaryotes, archaea and viruses. These communities have elaborate and highly structured biogeography that shapes metabolic exchange on a local scale and results from the diverse microenvironments present in the oral cavity. The oral microbiota also interfaces with the immune system of the human host and has an important role in not only the health of the oral cavity but also systemic health. In this Review, we highlight recent advances including novel insights into the biogeography of several oral niches at the species level, as well as the ecological role of candidate phyla radiation bacteria and non-bacterial members of the oral microbiome. In addition, we summarize the relationship between the oral microbiota and the pathology of oral diseases and systemic diseases. Together, these advances move the field towards a more holistic understanding of the oral microbiota and its role in health, which in turn opens the door to the study of novel preventive and therapeutic strategies.
Assuntos
Microbiota , Vírus , Humanos , Boca/microbiologia , Bactérias/genética , ArchaeaRESUMO
BACKGROUND: Porphyromonas gingivalis (hereafter "Pg") is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg. RESULTS: Here we systematically addressed the question of whether Pg are infected by phages-and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures. CONCLUSION: This work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly. Video Abstract.
Assuntos
Bacteriófagos , Doenças Periodontais , Humanos , Bacteriófagos/genética , Porphyromonas gingivalis/genética , Prófagos/genética , Sequência de BasesRESUMO
Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.
Assuntos
Bacteriófagos , Bacteriófagos/genética , Especificidade de HospedeiroRESUMO
Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in the wild remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that lytic interactions in environmental interaction networks (as observed in agar overlay) are sparse-with phage predator loads being low for most bacterial strains, and phages being host-strain-specific. Paradoxically, we also find that although overlap in killing is generally rare between tailed phages, recombination is common. Together, these results suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it is important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on lytic host range.
Assuntos
Bactérias/genética , Bactérias/virologia , Bacteriófagos/genética , Variação Genética , Genoma Viral , Especificidade de Hospedeiro , Modelos Biológicos , Nucleotídeos/metabolismo , Filogenia , Recombinases/metabolismo , Recombinação Genética/genética , Análise de Sequência de DNA , Vibrio/virologiaRESUMO
Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p = 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.
Assuntos
Bacteriófagos , Vírus , Bacteriófagos/genética , Códon/genética , Uso do Códon , RNA de Transferência/genética , RNA de Transferência/metabolismo , Vírus/genéticaRESUMO
Populations of the bacterium Vibrio cholerae consist of dozens of distinct lineages, with primarily (but not exclusively) members of the pandemic generating lineage capable of causing the diarrhoeal disease cholera. Assessing the composition and temporal dynamics of such populations requires extensive isolation efforts and thus only rarely covers large geographic areas or timeframes exhaustively. We developed a culture-independent amplicon sequencing strategy based on the protein-coding gene viuB (vibriobactin utilization) to study the structure of a V. cholerae population over the course of a summer. We show that the 26 co-occurring V. cholerae lineages continuously compete for limited space on nutrient-rich particles where only a few of them can grow to large numbers. Differential abundance of lineages between locations and size-fractions associated with a particle-attached or free-swimming lifestyle could reflect adaptation to various environmental niches. In particular, a major V. cholerae lineage occasionally grows to large numbers on particles but remain undetectable using isolation-based methods, indicating selective culturability for some members of the species. We thus demonstrate that isolation-based studies may not accurately reflect the structure and complex dynamics of V. cholerae populations and provide a scalable high-throughput method for both epidemiological and ecological approaches to studying this species.
Assuntos
Proteínas de Bactérias/genética , Catecóis/metabolismo , Cólera/microbiologia , Oxazóis/metabolismo , Vibrio cholerae/genética , Adaptação Fisiológica/genética , Humanos , Dinâmica Populacional , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
Natural selection shapes bacterial evolution in all environments. However, the extent to which commensal bacteria diversify and adapt within the human gut remains unclear. Here, we combine culture-based population genomics and metagenomics to investigate the within-microbiome evolution of Bacteroides fragilis. We find that intra-individual B. fragilis populations contain substantial de novo nucleotide and mobile element diversity, preserving years of within-person history. This history reveals multiple signatures of within-person adaptation, including parallel evolution in sixteen genes. Many of these genes are implicated in cell-envelope biosynthesis and polysaccharide utilization. Tracking evolutionary trajectories using near-daily metagenomic sampling, we find evidence for years-long coexistence in one subject despite adaptive dynamics. We used public metagenomes to investigate one adaptive mutation common in our cohort and found that it emerges frequently in Western, but not Chinese, microbiomes. Collectively, these results demonstrate that B. fragilis adapts within individual microbiomes, pointing to factors that promote long-term gut colonization.
Assuntos
Adaptação Biológica , Bacteroides fragilis/crescimento & desenvolvimento , Bacteroides fragilis/genética , Microbioma Gastrointestinal , Microbiota , Adulto , Feminino , Genética Populacional , Voluntários Saudáveis , Humanos , Masculino , Metagenômica , Mutação , Seleção Genética , Adulto JovemRESUMO
Viruses are highly discriminating in their interactions with host cells and are thought to play a major role in maintaining diversity of environmental microbes. However, large-scale ecological and genomic studies of co-occurring virus-host pairs, required to characterize the mechanistic and genomic foundations of virus-host interactions, are lacking. Here, we present the largest dataset of cultivated and sequenced co-occurring virus-host pairs that captures ecologically representative fine-scale diversity. Using the ubiquitous and ecologically diverse marine Vibrionaceae as a host platform, we isolate and sequence 251 dsDNA viruses and their hosts from three time points within a 93-day time-series study. The virus collection includes representatives of the three Caudovirales tailed virus morphotypes, a novel family of nontailed viruses, and the smallest (10,046 bp) and largest (348,911 bp) Vibrio virus genomes described. We provide general characterization and annotation of the viruses and describe read-mapping protocols to standardize genome presentation. The rich ecological and genomic contextualization of hosts and viruses make the Nahant Collection a unique platform for high-resolution studies of environmental virus-host infection networks.
Assuntos
Genoma Viral , Interações Hospedeiro-Patógeno , Vírus , Caudovirales , Ecologia , Vibrionaceae/virologia , Vírus/genéticaRESUMO
Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the Delftia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635-7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , DNA Viral/biossíntese , Timidina/química , Uridina/química , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/metabolismo , Genoma ViralRESUMO
The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.
Assuntos
Organismos Aquáticos/virologia , Bactérias/virologia , Vírus de DNA/classificação , Vírus de DNA/patogenicidade , Filogenia , Archaea/virologia , Viés , Proteínas do Capsídeo/metabolismo , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , Metagenômica , Vibrio/virologiaRESUMO
A universal tool in the culture-based study of bacterial viruses (bacteriophages, or phages) is the agar overlay, which is used in the isolation of new viruses, and in their quantification and purification. Here, simple optimizations that increase efficiency and throughput in agar overlay based isolation and cultivation of virus-host systems are presented. The agar overlay is streamlined to minimize steps and materials. Serial purification of viruses from viral colonies (plaques) is optimized to eliminate steps by combining purification by serial re-streaking with the optimized agar overlay approach. Finally, recommendations are made for efficient archival and storage of virus plaques. In sum, this work presents: â¢Tube-free Agar Overlays: rapid plaque assays with fewer steps and materialsâ¢Molten Streaking for Singles: rapid tube-free serial purification of virusesâ¢Archiving Plaques: saving virus purification for later.
RESUMO
With the overuse of antibiotics, many pathogens including Vibrio cholerae and Vibrio parahaemolyticus have evolved multidrug resistance making treatment more difficult. While understanding the mechanisms that underlie pathogenesis is crucial, knowledge of bacterial interactions of V. cholerae and V. parahaemolyticus could provide insight to their susceptibility outside of the human host. Based on previous work showing competition among environmental strains, we predict that marine-derived bacteria should inhibit Vibrio pathogens and may be a source of unique antibiotic compounds. We tested a collection of 3,456 environmental Vibrio isolates from diverse habitats against a panel of V. cholerae and V. parahaemolyticus, and identified 102 strains that inhibited the growth of these pathogens. Phylogenetic analysis revealed that 40 pathogen-inhibiting strains were unique at the hsp60 gene sequence while 62 of the isolates were identical suggesting clonal groups. Genomic comparisons of ten strains revealed diversity even between clonal isolates and were identified as being closely related to known Vibrio crassostreae, Vibrio splendidus, and Vibrio tasmaniensis strains. Further analysis revealed multiple biosynthetic gene clusters within all sequenced genomes that encoded secondary metabolites with potential antagonistic activity. Thus, environmental vibrios represent a source of compounds that inhibit Vibrio pathogens.
Assuntos
Antibiose , Microbiologia Ambiental , Vibrio cholerae/fisiologia , Vibrio parahaemolyticus/fisiologia , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Cólera/tratamento farmacológico , Cólera/microbiologia , Genes Bacterianos , Variação Genética , Genoma Bacteriano , Genômica/métodos , Genótipo , Humanos , Família Multigênica , Fenótipo , Filogenia , Seleção Genética , Vibrio/classificação , Vibrio/fisiologia , Vibrio cholerae/classificação , Vibrio cholerae/efeitos dos fármacos , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/efeitos dos fármacosRESUMO
UNLABELLED: Vibrio cholerae is a ubiquitous aquatic microbe in temperate and tropical coastal areas. It is a diverse species, with many isolates that are harmless to humans, while others are highly pathogenic. Most notable among them are strains belonging to the pandemic O1/O139 serogroup lineage, which contains the causative agents of cholera. The environmental selective regimes that led to this diversity are key to understanding how pathogens evolve in environmental reservoirs. A local population of V. cholerae and its close relative Vibrio metoecus from a coastal pond and lagoon system was extensively sampled during two consecutive months across four size fractions (480 isolates). In stark contrast to previous studies, the observed population was highly clonal, with 60% of V. cholerae isolates falling into one of five clonal complexes, which varied in abundance in the short temporal scale sampled. V. cholerae clonal complexes had significantly different distributions across size fractions and the two environments sampled, the pond and the lagoon. Sequencing the genomes of 20 isolates representing these five V. cholerae clonal complexes revealed different evolutionary trajectories, with considerable variations in gene content with potential ecological significance. Showing genotypic differentiation and differential spatial distribution, the dominant clonal complexes are likely ecologically divergent. Temporal variation in the relative abundance of these complexes suggests that transient blooms of specific clones could dominate local diversity. IMPORTANCE: Vibrio cholerae is commonly found in coastal areas worldwide, with only a single group of this bacterium capable of causing severe cholera outbreaks. However, the potential to evolve the ability to cause disease exists in many strains of this species in its aquatic reservoir. Understanding how pathogenic bacteria evolve requires the study of their natural environments. By extensive sampling in a geographically restricted location in the United States, we found that most cells of a V. cholerae population belong to only a small number of strains. Analysis of their genome composition and spatial distribution indicates differential environmental adaptations between these strains. Other strains exist in smaller numbers, and the population was found to be temporally varied. This suggests frequent bloom and collapse cycles on a time scale of weeks. These population dynamics make it possible that more virulent strains could stochastically rise to large numbers, allowing for infection to occur.
Assuntos
Variação Genética , Genótipo , Lagoas/microbiologia , Vibrio cholerae/classificação , Vibrio cholerae/genética , Evolução Molecular , Genes Bacterianos , Genoma Bacteriano , Análise de Sequência de DNA , Estados Unidos , Vibrio cholerae/isolamento & purificaçãoRESUMO
How reproducibly microbial populations assemble in the wild remains poorly understood. Here, we assess evidence for ecological specialization and predictability of fine-scale population structure and habitat association in coastal ocean Vibrionaceae across years. We compare Vibrionaceae lifestyles in the bacterioplankton (combinations of free-living, particle, or zooplankton associations) measured using the same sampling scheme in 2006 and 2009 to assess whether the same groups show the same environmental association year after year. This reveals complex dynamics with populations falling primarily into two categories: (i) nearly equally represented in each of the two samplings and (ii) highly skewed, often to an extent that they appear exclusive to one or the other sampling times. Importantly, populations recovered at the same abundance in both samplings occupied highly similar habitats suggesting predictable and robust environmental association while skewed abundances of some populations may be triggered by shifts in ecological conditions. The latter is supported by difference in the composition of large eukaryotic plankton between years, with samples in 2006 being dominated by copepods, and those in 2009 by diatoms. Overall, the comparison supports highly predictable population-habitat linkage but highlights the fact that complex, and often unmeasured, environmental dynamics in habitat occurrence may have strong effects on population dynamics.
Assuntos
Ecossistema , Plâncton/microbiologia , Vibrionaceae/fisiologia , Animais , Chaperonina 60/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Eucariotos/fisiologia , Filogenia , Vibrionaceae/genéticaRESUMO
Panulirus argus Virus 1 (PaV1) is a pathogenic virus that infects Caribbean spiny lobsters P. argus in the Florida Keys. We have developed a PCR detection assay for PaV1 for the purpose of studying the natural history of the virus and for monitoring the prevalence of infection. The detection of the virus in hemolymph and other tissues is based on the PCR amplification of a 499 bp product using specific primers designed from a cloned fragment of the PaV1 genome. The sensitivity limit for the assay was 1.2 fg of purified viral DNA. The PaV1 primers did not react with lobster DNA, oyster DNA, Ostreid Herpesvirus 1, or murine cytomegalovirus. Using this assay, we successfully followed the course of infection in lobsters inoculated with PaV1 and we detected infections in wild-caught lobsters from the Florida Keys. We have also established guidelines for interpreting infection results from the PCR assay for PaV1.