55 research outputs found

    Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

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    The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy

    Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa.

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    Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6 kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa

    Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity

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    BACKGROUND: Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. RESULTS: After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi(-) mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. CONCLUSIONS: This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-803) contains supplementary material, which is available to authorized users

    Comparative genomics and DNA methylation analysis of Pseudomonas aeruginosa clinical isolate PA3 by single-molecule real-time sequencing reveals new targets for antimicrobials

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    IntroductionPseudomonas aeruginosa (P.aeruginosa) is an important opportunistic pathogen with broad environmental adaptability and complex drug resistance. Single-molecule real-time (SMRT) sequencing technique has longer read-length sequences, more accuracy, and the ability to identify epigenetic DNA alterations.MethodsThis study applied SMRT technology to sequence a clinical strain P. aeruginosa PA3 to obtain its genome sequence and methylation modification information. Genomic, comparative, pan-genomic, and epigenetic analyses of PA3 were conducted.ResultsGeneral genome annotations of PA3 were discovered, as well as information about virulence factors, regulatory proteins (RPs), secreted proteins, type II toxin-antitoxin (TA) pairs, and genomic islands. A genome-wide comparison revealed that PA3 was comparable to other P. aeruginosa strains in terms of identity, but varied in areas of horizontal gene transfer (HGT). Phylogenetic analysis showed that PA3 was closely related to P. aeruginosa 60503 and P. aeruginosa 8380. P. aeruginosa's pan-genome consists of a core genome of roughly 4,300 genes and an accessory genome of at least 5,500 genes. The results of the epigenetic analysis identified one main methylation sites, N6-methyladenosine (m6A) and 1 motif (CATNNNNNNNTCCT/AGGANNNNNNNATG). 16 meaningful methylated sites were picked. Among these, purH, phaZ, and lexA are of great significance playing an important role in the drug resistance and biological environment adaptability of PA3, and the targeting of these genes may benefit further antibacterial studies.DisucssionThis study provided a detailed visualization and DNA methylation information of the PA3 genome and set a foundation for subsequent research into the molecular mechanism of DNA methyltransferase-controlled P. aeruginosa pathogenicity

    Antibacterial Activity of a Lytic Enzyme Encoded by Pseudomonas aeruginosa Double Stranded RNA Bacteriophage phiYY

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    Multidrug-resistant Pseudomonas aeruginosa is one of the most life-threatening pathogens for global health. In this regard, phage encoded lytic proteins, including endolysins and virion-associated peptidoglycan hydrolases (VAPGH), have been proposed as promising antimicrobial agents to treat P. aeruginosa. Most dsDNA phages use VAPGH to degrade peptidoglycan (PG) during infection, and endolysin to lyse the host cells at the end of lytic cycle. By contrast, dsRNA phage encodes only one lytic protein, which is located in the viral membrane to digest the PG during penetration, and also serves as an endolysin to release the phage. Currently, there are only seven sequenced dsRNA phages, and phiYY is the only one that infects human pathogen P. aeruginosa. In this study, dsRNA phage phiYY encoded lysin, named Ply17, was cloned and purified. Ply17 contains a PG-binding domain and a lysozyme-like-family domain. Ply17 exhibited a broad antibacterial activity against the outer membrane permeabilizer treated Gram-negative bacteria. The best lytic activity was achieved at 37°C, pH 7.5, in the presence of 0.5 mM EDTA. Moreover, it could effectively lyse Gram-positive bacteria directly, including Staphylococcus aureus. Therefore, dsRNA phage encoded Ply17 might be a promising new agent for treating multidrug-resistant pathogens

    Adaptation of Pseudomonas aeruginosa to Phage PaP1 Predation via O-Antigen Polymerase Mutation

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    Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O-antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior

    Characterization of spontaneous mutant phages.

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    <p><b>A</b>: Adsorption assay of bacteriophages JG004-m0 and JG004 to <i>P. aeruginosa</i> PA1 and PAO1. <b>B</b>: Gene organization of the region encoding tail proteins in JG004. The star in ORF84 indicates an A1959C mutation in ORF84 (2058 bp) in mutant phage JG004-m0. The alignment of the DNA and amino acid sequences of ORF84 between phage JG004 and five mutant phages indicates that the adenine (A) residue in phage JG004 changed to cytosine (C) in the mutant phages (shown in red).</p

    Characterization of recombinant phages.

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    <p><b>A</b>: PCR assay for confirmation of recombinant phages. <b>B</b>: Spot assay for testing the ability of PaP1, PaP1-rec1, and JG004 to infect PAO1 and PA1. <b>C</b>: Adsorption assay for testing the specificity of JG004, PaP1, and recombinant PaP1-rec1 in binding to <i>P. aeruginosa</i> strains PA1 and PAO1.</p

    Phages PaP1 and JG004 show different host specificities.

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    <p><b>A</b>: Spot assays for testing host specificity of phages PaP1 and JG004. <b>B</b>: Adsorption assay of bacteriophages PaP1 and JG004 to <i>P. aeruginosa</i> PA1 and PAO1. Percent adsorption of the phage was calculated as [(initial titer − residual titer) / initial titer] × 100%.</p
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