RESUMO
The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and â¼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding â¼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.
Assuntos
Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/virologia , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Animais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas , Pseudomonas aeruginosa/crescimento & desenvolvimento , RNA Bacteriano/análise , RNA Bacteriano/classificação , RNA Mensageiro/genética , RNA de Transferência/genética , RNA não Traduzido , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genéticaRESUMO
Bacterial viruses, or phage, are key members of natural microbial communities. Yet much research on bacterial-phage interactions has been conducted in liquid cultures involving single bacterial strains. Here we explored how bacterial diversity affects the success of lytic phage in structured communities. We infected a sensitive Pseudomonas aeruginosa strain PAO1 with a lytic phage Pseudomonas 352 in the presence versus absence of an insensitive P. aeruginosa strain PA14, in liquid culture versus colonies on agar. We found that both in liquid and in colonies, inter-strain competition reduced resistance evolution in the susceptible strain and decreased phage population size. However, while all sensitive bacteria died in liquid, bacteria in colonies could remain sensitive yet escape phage infection, due mainly to reduced growth in colony centers. In sum, spatial structure can protect bacteria against phage infection, while the presence of competing strains reduces the evolution of resistance to phage.
Assuntos
Biofilmes/crescimento & desenvolvimento , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Fagos de Pseudomonas/ultraestrutura , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/fisiologia , Especificidade da EspécieRESUMO
Invasive species populations periodically collapse from high to low abundance, sometimes even to extinction. Pathogens and the burden they place on invader immune systems have been hypothesised as a mechanism for these collapses. We examined the association of the bacterial pathogen (Pseudomonas spp.) and the viral community with immune gene expression in the globally invasive Argentine ant (Linepithema humile (Mayr)). RNA-seq analysis found evidence for 17 different viruses in Argentine ants from New Zealand, including three bacteriophages with one (Pseudomonas phage PS-1) likely to be attacking the bacterial host. Pathogen loads and prevalence varied immensely. Transcriptomic data showed that immune gene expression was consistent with respect to the viral classification of negative-sense, positive-sense and double-stranded RNA viruses. Genes that were the most strongly associated with the positive-sense RNA viruses such as the Linepithema humile virus 1 (LHUV-1) and the Deformed wing virus (DWV) were peptide recognition proteins assigned to the Toll and Imd pathways. We then used principal components analysis and regression modelling to determine how RT-qPCR derived immune gene expression levels were associated with viral and bacterial loads. Argentine ants mounted a substantial immune response to both Pseudomonas and LHUV-1 infections, involving almost all immune pathways. Other viruses including DWV and the Kashmir bee virus appeared to have much less immunological influence. Different pathogens were associated with varying immunological responses, which we hypothesize to interact with and influence the invasion dynamics of this species.
Assuntos
Formigas/imunologia , Imunidade Inata , Vírus de Insetos/patogenicidade , Fagos de Pseudomonas/patogenicidade , Pseudomonas/patogenicidade , Animais , Formigas/genética , Formigas/microbiologia , Formigas/virologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Espécies Introduzidas , Pseudomonas/virologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , TranscriptomaRESUMO
Pseudomonas aeruginosa (P. aeruginosa) infection has imposed a great threat to patients with cystic fibrosis. With the emergence of multidrug-resistant P. aeruginosa, developing an alternative anti-microbial strategy is indispensable and more urgent than ever. In this study, a lytic P. aeruginosa phage was isolated from the sewage of a hospital, and one protein was predicted as the depolymerase-like protein by genomic sequence analysis, it includes two catalytic regions, the Pectate lyase_3 super family and Glycosyl hydrolase_28 super family. Further analysis demonstrated that recombinant depolymerase-like protein degraded P. aeruginosa exopolysaccharide and enhanced bactericidal activity mediated by serum in vitro. Additionally, this protein disrupted host bacterial biofilms. All of these results showed that the phage-derived depolymerase-like protein has the potential to be developed into an anti-microbial agent that targets P. aeruginosa.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Pseudomonas/virologia , Fagos de Pseudomonas/genética , Pseudomonas aeruginosa/virologia , Resistência a Múltiplos Medicamentos/genética , Humanos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/patogenicidade , Esgotos/microbiologia , Esgotos/virologiaRESUMO
Like all viruses, bacteriophages heavily depend on their host's physiology for reproduction. Therefore, phages have evolved numerous proteins that influence the host metabolism to facilitate the infection process. Some of these proteins strongly perturb the host cell, ultimately leading to cell death. These growth-inhibitory phage proteins presumably target key metabolic processes, which may provide a basis for innovative phage-derived antibacterials. Unfortunately, most of these proteins are the so-called ORFans, since they have no known function or sequence homology to any other gene. We here describe a screening method for the identification of growth-inhibitory ORFans of bacteriophages infecting gram-negative bacteria (e.g., Pseudomonas aeruginosa), using the pUC18-mini-Tn7T-Lac vector system, which allows for stable single-copy integration of the phage ORFans in the Pseudomonas genome under the control of an IPTG-inducible promoter. Furthermore, we describe a method to examine the effect of the phage proteins in different hosts, using different vector copy numbers. Finally, we explain how to investigate the effect of ORFan expression on the host morphology using time-lapse microscopy.
Assuntos
Ensaios de Triagem em Larga Escala , Fagos de Pseudomonas/isolamento & purificação , Pseudomonas aeruginosa/genética , Proteínas Virais/metabolismo , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Fases de Leitura Aberta/genética , Filogenia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/virologia , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/metabolismo , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/virologia , Proteínas Virais/genéticaRESUMO
Nonmammalian infection models have been exploited to understand the various aspects of host-pathogen interactions and also provided innovative research platforms for identification of virulence factors, screening for antimicrobial hits, and evaluation of antimicroial efficacy. Here we describe a relatively straightforward protocol to assess the antibacterial efficacy of bacteriophages (phages) toward the opportunistic human pathogen, Pseudomonas aeruginosa, based on the systemic infection model using the fruit fly, Drosophila melanogaster. Since phages, unlike antibacterial chemicals, can be easily and sensitively enumerated by simple assays, it is also possible to address the pharmacokinetic properties of administered phages even in this small-scale infection model.
Assuntos
Terapia por Fagos/métodos , Infecções por Pseudomonas/terapia , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/virologia , Animais , Modelos Animais de Doenças , Drosophila melanogaster/microbiologia , Drosophila melanogaster/virologia , Interações Hospedeiro-Patógeno , Humanos , Infecções por Pseudomonas/virologia , Pseudomonas aeruginosa/patogenicidadeRESUMO
Psychrotrophic gram-negative Pseudomonas spp. represent a serious problem in the dairy industry as they can cause spoilage of milk and dairy products. Bacteriophages have moved into focus as promising biocontrol agents for such food spoilage bacteria. The virulent Siphoviridae phage PMBT14 was isolated on a mutant variant of P. fluorescens DSM 50090 challenged with an unrelated virulent P. fluorescens DSM 50090 Podoviridae phage (i.e., mutant strain DSM 50090R). PMBT14 has a 47,820-bp dsDNA genome with 76 predicted open reading frames (ORFs). Its genome shows no significant sequence similarity to that of known phages, suggesting that PMBT14 represents a novel phage. Phage PMBT14 could be a promising biocontrol agent for P. fluorescens in milk or dairy foods.
Assuntos
Genoma Viral , Lisogenia/fisiologia , Fagos de Pseudomonas/genética , Pseudomonas fluorescens/virologia , Siphoviridae/genética , Proteínas Virais/genética , Agentes de Controle Biológico , Mapeamento Cromossômico , DNA/genética , DNA/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Laticínios/microbiologia , Microbiologia de Alimentos , Ontologia Genética , Tamanho do Genoma , Humanos , Anotação de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Fagos de Pseudomonas/classificação , Fagos de Pseudomonas/patogenicidade , Fagos de Pseudomonas/ultraestrutura , Análise de Sequência de DNA , Siphoviridae/classificação , Siphoviridae/metabolismo , Proteínas Virais/metabolismoRESUMO
Phage preparations used for phage therapy may have not only direct antibacterial action but also immunomodulating effects mediated by phages themselves as well as by bacterial antigens. Therefore phage application in patients with immune disorders, and especially with autoimmune diseases, requires special attention. The aim of this study was to investigate the effect of phage lysates (staphylococcal phages A3/R, phi200, and MS-1 cocktail, enterococcal phage 15/P, Pseudomonas phage 119x, and E. coli T4 phage) as well as purified T4 phage on the course of murine collagen-induced arthritis (CIA), commonly used as an animal model of rheumatoid arthritis. Intraperitoneal application of phage lysates or purified T4 phage did not aggravate the course of autoimmune joint disease. Moreover, although endotoxins are known to potentiate CIA, the systemic administration of phage lysate of Pseudomonas aeruginosa, which contains debris of this Gram-negative bacillus, did not significantly influence CIA although the sonicate of the corresponding bacterial strain did. Interestingly, a purified T4 phage revealed some anti-inflammatory activity when applied under the therapeutic scheme. Our preliminary results do not suggest that phages may aggravate the symptoms of rheumatoid arthritis. In contrast T4 phage may even exert an immunosuppressive effect.
Assuntos
Artrite Experimental/terapia , Doenças Autoimunes/imunologia , Bacteriófago T4/imunologia , Terapia por Fagos/métodos , Animais , Artrite Experimental/complicações , Artrite Experimental/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/virologia , Bacteriófago T4/patogenicidade , Modelos Animais de Doenças , Escherichia coli/imunologia , Escherichia coli/virologia , Humanos , Imunomodulação/imunologia , Camundongos , Terapia por Fagos/efeitos adversos , Fagos de Pseudomonas/imunologia , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/virologia , Fagos de Staphylococcus/imunologia , Fagos de Staphylococcus/patogenicidadeRESUMO
Bacteriophage PAXYB1 was recently isolated from wastewater samples. This phage was chosen based on its lytic properties against clinical isolates of Pseudomonas aeruginosa (P. aeruginosa). In the present study, characterized PAXYB1, clarified its morphological and lytic properties, and analyzed its complete genome sequence. Based on the morphology of PAXYB1, it is a Podoviridae. The linear GC-rich (62.29%) double-stranded DNA genome of PAXYB1 is 43,337 bp including direct terminal repeats (DTRs) of 468 bp. It contains 60 open reading frames (ORFs) that are all encoded within the same strand. We also showed that PAXYB1 is a virulent phage and a new member of the phiKMV-like phages genus. Twenty-eight out of sixty predicted gene products (gps) showed significant homology to proteins of known function, which were confirmed by analyzing the structural proteome. Altogether, our work identified a novel lytic bacteriophage that lyses P. aeruginosa PAO1 and efficiently infects and kills several clinical isolates of P. aeruginosa. This phage has potential for development as a biological disinfectant to control P. aeruginosa infections.
Assuntos
Pseudomonas aeruginosa/virologia , Genes Virais/genética , Genoma Viral/genética , Genômica/métodos , Fases de Leitura Aberta/genética , Podoviridae/genética , Podoviridae/patogenicidade , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/patogenicidade , Análise de Sequência de DNARESUMO
A virulent bacteriophage highly specific to Pseudomonas aeruginosa (P. aeruginosa) was isolated from hospital sewage using a lambda bacteriophage isolation protocol. The bacteriophage, named as PAP1, was used to functionalize tosyl-activated magnetic beads to establish a bacteriophage-affinity strategy for separation and detection of viable P. aeruginosa. Recognition of the target bacteria by tail fibers and baseplate of the bacteriophage led to capture of P. aeruginosa onto the magnetic beads. After a replication cycle of about 100 min, the progenies lysed the target bacteria and released the intracellular adenosine triphosphate. Subsequently, firefly luciferase-adenosine triphosphate bioluminescence system was used to quantitate the amount of P. aeruginosa. This bacteriophage-affinity strategy for viable P. aeruginosa detection showed a linear range of 6.0 × 102 to 3.0 × 105 CFU mL-1, with a detection limit of 2.0 × 102 CFU mL-1. The whole process for separation and detection could be completed after bacteria capture, bacteriophage replication, and bacteria lysis within 2 h. Since the isolated bacteriophage recognized the target bacteria with very high specificity, the proposed strategy did not show any signal response to all of the tested interfering bacteria. Furthermore, it excluded the interference from inactivated P. aeruginosa because the bacteriophage could replicate only in viable cells. The proposed strategy had been applied for detection of P. aeruginosa in glucose injection, human urine, and rat plasma. In the further work, this facile bacteriophage-affinity strategy could be extended for detection of other pathogens by utilizing virulent bacteriophage specific to other targets.
Assuntos
Técnicas Biossensoriais/métodos , Separação Imunomagnética/métodos , Fagos de Pseudomonas/fisiologia , Pseudomonas aeruginosa/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Animais , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Limite de Detecção , Luminescência , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fagos de Pseudomonas/patogenicidade , Fagos de Pseudomonas/ultraestrutura , Pseudomonas aeruginosa/metabolismo , Ratos Sprague-Dawley , VirulênciaRESUMO
Resource availability can affect the coevolutionary dynamics between host and parasites, shaping communities and hence ecosystem function. A key finding from theoretical and in vitro studies is that host resistance evolves to greater levels with increased resources, but the relevance to natural communities is less clear. We took two complementary approaches to investigate the effect of resource availability on the evolution of bacterial resistance to phages in soil. First, we measured the resistance and infectivity of natural communities of soil bacteria and phage in the presence and absence of nutrient-providing plants. Second, we followed the real-time coevolution between defined bacteria and phage populations with resource availability manipulated by the addition or not of an artificial plant root exudate. Increased resource availability resulted in increases in bacterial resistance to phages, but without a concomitant increase in phage infectivity. These results suggest that phages may have a reduced impact on the control of bacterial densities and community composition in stable, high resource environments.
Assuntos
Evolução Molecular , Fagos de Pseudomonas/genética , Pseudomonas fluorescens/genética , Seleção Genética , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Infecções Bacterianas/virologia , Humanos , Fagos de Pseudomonas/patogenicidade , Pseudomonas fluorescens/virologia , Microbiologia do SoloRESUMO
The sensitivity of 512 newly isolated Pseudomonas aeruginosa clinical strains to six classes of anti-microbial preparations has been studied. Antibiotic-resistant strains were selected and genotyped. Three new virulent bacteriophages of the families Myoviridae and Podoviridae were isolated against these strains. The parameters of the intracellular phage development cycle were established, and the influence of inactivating factors (temperature, pH, and UV exposure) on phage viability was studied. The molecular weight of the phage genome was determined. Phage DNA restriction analysis and polyacrylamide gel electrophoresis in the presence of envelope protein SDS were carried out. The plating efficacy of phages on 28 genetically distant antibiotic-resistant P. aeruginosa strains was studied. It was established that 26 of them were lysed by phages with a high efficacy. The range of antibacterial action of the studied phages and their mixtures on 427 multi-drug-resistant clinical isolates was assessed. It is shown that including these phages in one multicomponent preparation enhanced their lytic activity.
Assuntos
Genoma Viral , Myoviridae/patogenicidade , Podoviridae/patogenicidade , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/virologia , Farmacorresistência Bacteriana Múltipla , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Lisogenia , Tipagem Molecular , Peso Molecular , Myoviridae/classificação , Myoviridae/genética , Myoviridae/isolamento & purificação , Filogenia , Podoviridae/classificação , Podoviridae/genética , Podoviridae/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Fagos de Pseudomonas/classificação , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/isolamento & purificação , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/isolamento & purificação , Temperatura , Raios Ultravioleta , VirulênciaRESUMO
We still know very little about how the environment influences coevolutionary dynamics. Here, we investigated both theoretically and empirically how nutrient availability affects the relative extent of escalation of resistance and infectivity (arms race dynamic; ARD) and fluctuating selection (fluctuating selection dynamic; FSD) in experimentally coevolving populations of bacteria and viruses. By comparing interactions between clones of bacteria and viruses both within- and between-time points, we show that increasing nutrient availability resulted in coevolution shifting from FSD, with fluctuations in average infectivity and resistance ranges over time, to ARD. Our model shows that range fluctuations with lower nutrient availability can be explained both by elevated costs of resistance (a direct effect of nutrient availability), and reduced benefits of resistance when population sizes of hosts and parasites are lower (an indirect effect). Nutrient availability can therefore predictably and generally affect qualitative coevolutionary dynamics by both direct and indirect (mediated through ecological feedbacks) effects on costs of resistance.
Assuntos
Evolução Biológica , Fagos de Pseudomonas/genética , Pseudomonas fluorescens/genética , Modelos Biológicos , Dinâmica Populacional , Fagos de Pseudomonas/patogenicidade , Pseudomonas fluorescens/virologia , Seleção GenéticaRESUMO
The potential of bacteriophage therapy to treat infections caused by antibiotic-resistant bacteria has now been well established using various animal models. While numerous newly isolated bacteriophages have been claimed to be potential therapeutic candidates on the basis of in vitro observations, the parameters used to guide their choice among billions of available bacteriophages are still not clearly defined. We made use of a mouse lung infection model and a bioluminescent strain of Pseudomonas aeruginosa to compare the activities in vitro and in vivo of a set of nine different bacteriophages (PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5, CHA_P1, LBL3, LUZ19, and PhiKZ). For seven bacteriophages, a good correlation was found between in vitro and in vivo activity. While the remaining two bacteriophages were active in vitro, they were not sufficiently active in vivo under similar conditions to rescue infected animals. Based on the bioluminescence recorded at 2 and 8 h postinfection, we also define for the first time a reliable index to predict treatment efficacy. Our results showed that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting a personalized approach favoring an optimal treatment.
Assuntos
Terapias Complementares/métodos , Pneumonia Bacteriana/terapia , Infecções por Pseudomonas/terapia , Fagos de Pseudomonas/crescimento & desenvolvimento , Pseudomonas aeruginosa/virologia , Animais , Bioensaio , Modelos Animais de Doenças , Genes Reporter , Medições Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/microbiologia , Valor Preditivo dos Testes , Infecções por Pseudomonas/microbiologia , Fagos de Pseudomonas/patogenicidade , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Resultado do Tratamento , Ensaio de Placa ViralRESUMO
Although pervasive, the impact of temporal environmental heterogeneity on coevolutionary processes is poorly understood. Productivity is a key temporally heterogeneous variable, and increasing productivity has been shown to increase rates of antagonistic arms race coevolution, and lead to the evolution of more broadly resistant hosts and more broadly infectious parasites. We investigated the effects of the grain of environmental heterogeneity, in terms of fluctuations in productivity, on bacteria-phage coevolution. Our findings demonstrate that environmental heterogeneity could constrain antagonistic coevolution, but that its effect was dependent upon the grain of heterogeneity, such that both the rate and extent of coevolution were most strongly limited in fine-grained, rapidly fluctuating heterogeneous environments. We further demonstrate that rapid environmental fluctuations were likely to have impeded selective sweeps of resistance alleles, which occurred over longer durations than the fastest, but not the slowest, frequency of fluctuations used. Taken together our results suggest that fine-grained environmental heterogeneity constrained the coevolutionary arms race by impeding selective sweeps.
Assuntos
Evolução Biológica , Fagos de Pseudomonas/fisiologia , Fagos de Pseudomonas/patogenicidade , Pseudomonas fluorescens/fisiologia , Pseudomonas fluorescens/virologia , Seleção Genética , Meio Ambiente , Fatores de TempoRESUMO
The broad-host-range lytic Pseudomonas phage Φ-S1 possess a 40,192 bp double-stranded DNA (dsDNA) genome of 47 open reading frames (ORFs) and belongs to the family Podoviridae, subfamily Autographivirinae, genus T7likevirus.
Assuntos
Podoviridae/genética , Fagos de Pseudomonas/genética , Sequência de Bases , DNA Viral/genética , Genoma Viral , Especificidade de Hospedeiro , Dados de Sequência Molecular , Podoviridae/classificação , Podoviridae/patogenicidade , Fagos de Pseudomonas/classificação , Fagos de Pseudomonas/patogenicidade , Pseudomonas fluorescens/virologia , Esgotos/virologiaRESUMO
Coinfection with multiple parasite genotypes [multiplicity of infection (MOI)] creates within-host competition and opportunities for parasite recombination and is therefore predicted to be important for both parasite and host evolution. We tested for a difference in the infectivity of viral parasites (lytic phage Φ2) and resistance of their bacterial hosts (Pseudomonas fluorescens SBW25) under both high and low MOI during coevolution in laboratory microcosms. Results show that MOI has no effect on infectivity and resistance evolution during coevolution over â¼80 generations of host growth, and this is true when the experiment is initiated with wild-type viruses and hosts, or with viruses and hosts that have already been coevolving for â¼330 generations. This suggests that MOI does not have a net effect of accelerating parasite adaptation to hosts through recombination, or slowing adaptation to hosts through between-parasite conflict in this system.
Assuntos
Fagos de Pseudomonas/genética , Pseudomonas fluorescens/virologia , Adaptação Fisiológica/genética , Evolução Molecular , Fenótipo , Fagos de Pseudomonas/patogenicidade , Recombinação GenéticaRESUMO
Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P. aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P. aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P. aeruginosa in the environment.
Assuntos
Fagos de Pseudomonas/classificação , Pseudomonas aeruginosa/virologia , Impressões Digitais de DNA , DNA Viral/química , Especificidade de Hospedeiro , Myoviridae/classificação , Myoviridae/genética , Podoviridae/classificação , Podoviridae/genética , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/isolamento & purificação , Fagos de Pseudomonas/patogenicidade , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Parasites can promote diversity by mediating coexistence between a poorer and superior competitor, if the superior competitor is more susceptible to parasitism. However, hosts and parasites frequently undergo antagonistic coevolution. This process may result in the accumulation of pleiotropic fitness costs associated with host resistance, and could breakdown coexistence. We experimentally investigated parasite-mediated coexistence of two genotypes of the bacterium Pseudomonas fluorescens, where one genotype underwent coevolution with a parasite (a virulent bacteriophage), whereas the other genotype was resistant to the evolving phages at all time points, but a poorer competitor. In the absence of phages, the resistant genotype was rapidly driven extinct in all populations. In the presence of the phages, the resistant genotype persisted in four of six populations and eventually reached higher frequencies than the sensitive genotype. The coevolving genotype showed a reduction in the growth rate, consistent with a cost of resistance, which may be responsible for a decline in its relative fitness. These results demonstrate that the stability of parasite-mediated coexistence of resistant and susceptible species or genotypes is likely to be affected if parasites and susceptible hosts coevolve.
Assuntos
Evolução Biológica , Interações Hospedeiro-Parasita/fisiologia , Fagos de Pseudomonas/fisiologia , Fagos de Pseudomonas/patogenicidade , Pseudomonas fluorescens/virologia , Genótipo , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/crescimento & desenvolvimentoRESUMO
Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.