| Pseudomonas aeruginosa(P. aeruginosa) is a gram-negative opportunistic pathogen. P. aeruginosa have a remarkable ability to adapt to changing environments and a wide distribution in the biosphere. The infection of P. aeruginosa is found in pulmonary tract, burns and wounds. Recently, extensive application of antibiotic accelerates the emergence of resistance to existing antibiotics and multidrug-resistant bacteria that are difficult to treat. The widespread bacterial resistance to antibiotics poses a serious threat to public health, such as the multidrug-resistant “ESKAPE” pathogens(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species) that cause the majority of nosocomial infection[1]. Among these pathogens, some strains are even resistant to all currently available antibiotics[2]. The genome of P. aeruginosa, contaning virous drug resistance genes, is one of the largest bacterial genomes. Since traditional antibiotic discovery efforts have failed to keep up with the evolution of bacterial resistance, the treatment of multidrug-resistant P. aeruginosa become very hard. Bacteriophages(phages) are obligate intracellular parasites[3]. Since the first discovery by in 1915, bactriaphage was used to treat the infection of P. aeruginosa and showed with a good therapeutic effect. There are approximately 1031 phages(ten times the number of bacteria) in the biosphere, which provides a huge resource pool for exploring potential antibacterial compounds. However, there were only about 1,400 phages have been sequenced currently and most of the phage genes had no annotations. Our knowledge about phage genomics and gene function is just the tip of the iceberg. Lack of knowledge blocks the usage of the bacteriophage for us. Therefore, with the aim to use the bacteriophage as a weapon to defeat the multidrug-resistant bacteria, the deep insights into phage-host interactions and the phage encoded proteins were badly needed.In this work, we investigated the genome-wide interactions between phage Pa P3 with its host-P. aeruginosa PA3 according to the one-step growth curve of Pa P3. The co-expression networks of phage and host was constructed on the basis of microarray dataset. Furthermore, a bacteria growth inhibitor encoded by orf70.1 of phage Pa P3 was indentified. The combination of mutifaced analysis including: GST-pull down, microarray analysis, RT-q PCR, NMR spectroscopy and phenotypic analysis was performed to invetigate the function of orf70.1. The main contents of this study are showed as follows:The global impact of phage Pa P3 infection on host bacterial transcriptome: we investigate genome-wide interactions of P. aeruginosa and its temperate phage Pa P3 at five time points during phage infection. Compared to the uninfected host, the impact of Pa P3 on host P. aeruginosa gene expression was mainly in midlle stage of phage infection. Globally, there were 38% DEGs of P. aeruginosa comparing the infected samples to the uninfected samples and 98% DEGs were down-regulated. Functional analysis of the DEGs revealed infection-stage-dependent pathway communications.Gene interaction networks between phage Pa P3 and P. aeruginosa: According to the microarray analysis, gene co-expression analysis based on pearson correlation coefficient was used to uncover the correlations between phage and host transcriptional regulators(TRs) and construct gene interaction networks between Pa P3 and host at each stage of infection as well as a merged network of gene co-expression with five time points. We drew the following conclusion: 1) The correlations between phage and host TRs were mostly negative. Specific gene products of Pa P3 might target host TRs and lead to differentially expressed downstream genes. 2) the genes that appeared in one sub-network might share similar biological processes, gene co-expression was used to predict the biological function of several unknown Pa P3 genes. 3) The early genes of phage Pa P3 were in the center of the regulatory network. The function of amino acid biosynthesis and transport of small molecules could be the hotspots specific to the regulation of phage.Re-annotation of the phage Pa P3 genes: The combination of six platforms for gene prediction was used to re-annotate the phage Pa P3 genes. RT-q PCR analysis was further performed to validate the prediction. Finally, the Pa P3 genes were increased from previous 71 genes to current 126 genes.The screening of antimicrobial protein from phage Pa P3: In our previous work, two gene products encoded by phage Pa P3 showed interactions with host proteins. One product, named gp70.1(gene product, gp) encoded by orf70.1 was identified to have inhibitory effect on bacterial growth. P. aeruginosa PA3 containing gp70.1 appeared a needle-like conoly on solid medium and a delayed growth in liquit culture. Features of the bacteria growth inhibitor-gp70.1: Bioinformatics analysis and Blast P against non-redundant protein sequences showed that no putative conserved domains were detected in gp70.1. The molecular weight of gp70.1 was approximately 13 kd and consistent with the prediction according to SDS-PAGE for the purified gp70.1. Furtheremore, polyclonal antibody against gp70.1 was prepared, by which the GST-pull down assay showed the direct binding of gp70.1 with Rpo S in vitro.The influence of gp70.1 on Rpo S: The RNA polymerase sigma factor Rpo S was a global transcriptional regulator in P. aeruginas. The direct binding of gp70.1 to Rpo S was shown to cause the shutoff of Rpo S-regulated stress response, biofilm and virulence.Mutifeced analysis for the influence of gp70.1 on P. aeruginosa: The combination of multifaceted analysis including microarray assay, RT-q PCR, nuclear magnetic resonance(NMR) spectroscopy and phenotype experiments were performed to investigate the effects of gp70.1 on the P. aeruginosa. A total of 178 genes of P. aeruginosa mainly involving in extracellular function and metabolism were differential expressed in the presence of gp70.1 for three examined time points. Furthermore, our results indicated that gp70.1 had the extensive impact on the extracellular phenotype of P. aeruginosa, such as motility, pyocyanin, extracellular protease, polysaccharide, and cellulase. For the metabolism of P. aeruginosa, the main effect of gp70.1 was the reduction of amino acids consumption.In conclusion, this work uncovered the influence of phage Pa P3 infection on the transcritome of P. aeruginosa PA3 and construct a co-expression network of phage-host interactions at genomic level. This provided a base for the subsequent screening for bacteria growth inhibors from phage and further insight into phage-host interactions. Moreover, based on the re-annotation of the genes of phage Pa P3, a novel phage protein with a growth inhibitory effect to bacteria as well as its cellular target were identified. |
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