Shuguang Lu

Genomic and Proteomic Analyses of the Terminally Redundant Genome of the Pseudomonas aeruginosa Phage PaP1: Establishment of Genus PaP1-Like Phages

We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68–70 nm and a contractile tail with a length of 138–140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp) with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs). Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa.

Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa

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.

Anti-obesity Effect of Capsaicin in Mice Fed with High-Fat Diet Is Associated with an Increase in Population of the Gut Bacterium Akkermansia muciniphila

Capsaicin (CAP) reduces body weight mainly through activation of transient receptor potential vanilloid 1 (TRPV1) cation channel. However, recent evidence indicates that the gut microbiota influences many physiological processes in host and might provoke obesity. This study determined whether the anti-obesity effect of CAP is related to the changes in gut microbiota. C57BL/6 mice were fed either with high-fat diet (HFD) or HFD with CAP (HFD-CAP) for 9 weeks. We observed a significantly reduced weight gain and improved glucose tolerance in HFD-CAP-fed mice compared with HFD-fed mice. 16S rRNA gene sequencing results showed a decrease of phylum Proteobacteria in HFD-CAP-fed mice. In addition, HFD-CAP-fed mice showed a higher abundance of Akkermansia muciniphila, a mucin-degrading bacterium with beneficial effects on host metabolism. Further studies found that CAP directly up-regulates the expression of Mucin 2 gene Muc2 and antimicrobial protein gene Reg3g in the intestine. These data suggest that the anti-obesity effect of CAP is associated with a modest modulation of the gut microbiota.

Global Transcriptomic Analysis of Interactions between Pseudomonas aeruginosa and Bacteriophage PaP3.

The interactions between Bacteriophage (phage) and host bacteria are widespread in nature and influences of phage replication on the host cells are complex and extensive. Here, we investigate genome-wide interactions of Pseudomonas aeruginosa (P. aeruginosa) and its temperate phage PaP3 at five time points during phage infection. Compared to the uninfected host, 38% (2160/5633) genes of phage-infected host were identified as differentially expressed genes (DEGs). Functional analysis of the repressed DEGs revealed infection-stage-dependent pathway communications. Based on gene co-expression analysis, most PaP3 middle genes were predicted to have negative impact on host transcriptional regulators. Sub-network enrichment analysis revealed that adjacent genes of PaP3 interacted with the same host genes and might possess similar functions. Finally, our results suggested that during the whole infection stage, the early genes of PaP3 had stronger regulatory role in host gene expression than middle and late genes, while the host genes involved amino acid metabolism were the most “vulnerable” targets of these phage genes. This work provides the basis for understanding survival mechanisms of parasites and host, and seeking phage gene products that could potentially be used in anti-bacterial infection.

Identification and Characterization of the HicAB Toxin-Antitoxin System in the Opportunistic Pathogen Pseudomonas aeruginosa

Toxin-antitoxin (TA) systems are small genetic modules that are widely distributed in the genomes of bacteria and archaea and have been proposed to fulfill numerous functions. Here, we describe the identification and characterization of a type II TA system, comprising the hicAB locus in the human opportunistic pathogen Pseudomonas aeruginosa. The hicAB locus consists of genes hicA and hicB encoding a toxin and its cognate antitoxin, respectively. BLAST analysis revealed that hicAB is prevalent in approximately 36% of P. aeruginosa strains and locates in the same genomic region. RT-PCR demonstrated that hicAB forms a bicistronic operon that is cotranscribed under normal growth conditions. Overproduction of HicA inhibited the growth of Escherichia coli, and this effect could be counteracted by co-expression of HicB. The Escherichia coli kill/rescue assay showed that the effect of HicA is bacteriostatic, rather than bactericidal. Deletion of hicAB had no effect on the biofilm formation and virulence of P. aeruginosa in a mice infection model. Collectively, this study presents the first characterization of the HicAB system in the opportunistic pathogen P. aeruginosa.

The chromosomal SezAT toxin–antitoxin system promotes the maintenance of the SsPI‐1 pathogenicity island in epidemic Streptococcus suis

Streptococcus suis has emerged as a causative agent of human meningitis and streptococcal toxic shock syndrome over the last years. The high pathogenicity of S. suis may be due in part to a laterally acquired pathogenicity island (renamed SsPI‐1), which can spontaneously excise and transfer to recipients. Cells harboring excised SsPI‐1 can potentially lose this island if cell division occurs prior to its reintegration; however, attempts to cure SsPI‐1 from the host cells have been unsuccessful. Here, we report that an SsPI‐1‐borne Epsilon/Zeta toxin–antitoxin system (designated SezAT) promotes SsPI‐1 stability in bacterial populations. The sezAT locus consists of two closely linked sezT and sezA genes encoding a toxin and its cognate antitoxin, respectively. Overproduction of SezT induces a bactericidal effect that can be neutralized by co‐expression of SezA, but not by its later action. When devoid of a functional SezAT system, large‐scale deletion of SsPI‐1 is straightforward. Thus, SezAT serves to ensure inheritance of SsPI‐1 during cell division, which may explain the persistence of epidemic S. suis. This report presents the first functional characterization of TA loci in S. suis, and the first biochemical evidence for the adaptive significance of the Epsilon/Zeta system in the evolution of pathogen virulence.

Complete Genome Sequence of Pseudomonas aeruginosa PA1, Isolated from a Patient with a Respiratory Tract Infection

ABSTRACT We report the 6,498,072-bp complete genome sequence of Pseudomonas aeruginosa PA1, which was isolated from a patient with a respiratory tract infection in Chongqing, Peoples Republic of China. Whole-genome sequencing was performed using single-molecule real-time (SMRT) technology, and de novo assembly revealed a single contig with 396-fold sequence coverage.

Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa

Bacteriophages (phages) are widely distributed in the biosphere and play a key role in modulating microbial ecology in the soil, ocean, and humans. Although the role of DNA bacteriophages is well described, the biology of RNA bacteriophages is poorly understood. More than 1900 phage genomes are currently deposited in NCBI, but only 6 dsRNA bacteriophages and 12 ssRNA bacteriophages genome sequences are reported. The 6 dsRNA bacteriophages were isolated from legume samples or lakes with Pseudomonas syringae as the host. Here, we report the first Pseudomonas aeruginosa phage phiYY with a three-segmented dsRNA genome. phiYY was isolated from hospital sewage in China with the clinical P. aeruginosa strain, PAO38, as a host. Moreover, the dsRNA phage phiYY has a broad host range, which infects 99 out of 233 clinical P. aeruginosa strains isolated from four provinces in China. This work presented a detailed characterization of the dsRNA bacteriophage infecting P. aeruginosa.

Characterization and Comparative Genomic Analyses of Pseudomonas aeruginosa Phage PaoP5: New Members Assigned to PAK_P1-like Viruses

As a potential alternative to antibiotics, phages can be used to treat multi-drug resistant bacteria. As such, the biological characteristics of phages should be investigated to utilize them as effective antimicrobial agents. In this study, phage PaoP5, a lytic virus that infects Pseudomonas aeruginosa PAO1, was isolated and genomically characterized. PaoP5 comprises an icosahedral head with an apex diameter of 69 nm and a contractile tail with a length of 120 nm. The PaoP5 genome is a linear dsDNA molecule containing 93,464 base pairs (bp) with 49.51% G + C content of 11 tRNA genes and a 1,200 bp terminal redundancy. A total of 176 protein-coding genes were predicted in the PaoP5 genome. Nine PaoP5 structural proteins were identified. Three hypothetical proteins were determined as structural. Comparative genomic analyses revealed that seven new Pseudomonas phages, namely, PaoP5, K8, C11, vB_PaeM_C2-10_Ab02, vB_PaeM_C2-10_Ab08, vB_PaeM_C2-10_Ab10, and vB_PaeM_C2-10_Ab15, were similar to PAK_P1-like viruses. Phylogenetic and pan-genome analyses suggested that the new phages should be assigned to PAK_P1-like viruses, which possess approximately 100 core genes and 150 accessory genes. This work presents a detailed and comparative analysis of PaoP5 to enhance our understanding of phage biology.

Genomic analyses of multidrug resistant Pseudomonas aeruginosa PA1 resequenced by single-molecule real-time sequencing

As a third-generation sequencing (TGS) method, single-molecule real-time (SMRT) technology provides long read length, and it is well suited for resequencing projects and de novo assembly. In the present study, Pseudomonas aeruginosa PA1 was characterized and resequenced using SMRT technology. PA1 was also subjected to genomic, comparative and pan-genomic analyses. The multidrug resistant strain PA1 possesses a 6,498,072 bp genome and a sequence type of ST-782. The genome of PA1 was also visualized, and the results revealed the details of general genome annotations, virulence factors, regulatory proteins (RPs), secretion system proteins, type II toxin–antitoxin (T–A) pairs and genomic islands. Whole genome comparison analysis suggested that PA1 exhibits similarity to other P. aeruginosa strains but differs in terms of horizontal gene transfer (HGT) regions, such as prophages and genomic islands. Phylogenetic analyses based on 16S rRNA sequences demonstrated that PA1 is closely related to PAO1, and P. aeruginosa strains can be divided into two main groups. The pan-genome of P. aeruginosa consists of a core genome of approximately 4,000 genes and an accessory genome of at least 6,600 genes. The present study presented a detailed, visualized and comparative analysis of the PA1 genome, to enhance our understanding of this notorious pathogen.