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• W3048355891 abstract "•The infection of dsRNA bacteriophage has a modest impact on host gene expression•Several differentially expressed host genes are essential for dsRNA phage life cycle•Anti-oxidative genes are highly expressed to support dsRNA phage infection Bacteriophage phiYY is currently the only double-stranded RNA (dsRNA) phage that infects Pseudomonas aeruginosa and is a potential candidate for phage therapy. Here we applied RNA-seq to investigate the lytic cycle of phiYY infecting P. aeruginosa strain PAO1r. About 12.45% (651/5,229) of the host genes were determined to be differentially expressed genes. Moreover, oxidative stress response genes katB and ahpB are upregulated 64- to 128-fold after phage infection, and the single deletion of each gene blocked phiYY infection, indicating that phiYY is extremely sensitive to oxidative stress. On the contrary, another upregulated gene PA0800 might constrain phage infection, because the deletion of PA0800 resulted in a 3.5-fold increase of the efficiency of plating. Our study highlights a complicated dsRNA phage-phage global interaction and raises new questions toward the host defense mechanisms against dsRNA phage and dsRNA phage-encoded hijacking mechanisms. Bacteriophage phiYY is currently the only double-stranded RNA (dsRNA) phage that infects Pseudomonas aeruginosa and is a potential candidate for phage therapy. Here we applied RNA-seq to investigate the lytic cycle of phiYY infecting P. aeruginosa strain PAO1r. About 12.45% (651/5,229) of the host genes were determined to be differentially expressed genes. Moreover, oxidative stress response genes katB and ahpB are upregulated 64- to 128-fold after phage infection, and the single deletion of each gene blocked phiYY infection, indicating that phiYY is extremely sensitive to oxidative stress. On the contrary, another upregulated gene PA0800 might constrain phage infection, because the deletion of PA0800 resulted in a 3.5-fold increase of the efficiency of plating. Our study highlights a complicated dsRNA phage-phage global interaction and raises new questions toward the host defense mechanisms against dsRNA phage and dsRNA phage-encoded hijacking mechanisms. Bacteriophage phiYY is a recently identified member of Cystoviridae (Yang et al., 2016Yang Y.H. Lu S.G. Shen W. Zhao X. Shen M.Y. Tan Y.L. Li G. Li M. Wang J. Hu F.Q. Le S. Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa.Sci. Rep. 2016; 9: 38795Crossref Scopus (34) Google Scholar), which have genomes consisting of three double-stranded RNA molecules, L, M, and S. Phage Φ6 was the first identified member of this family isolated in 1973 (Vidaver et al., 1973Vidaver A.K. Koski R.K. Van Etten J.L. Bacteriophage phi6: a lipid-containing virus of Pseudomonas phaseolicola.J. Virol. 1973; 11: 799-805Crossref PubMed Google Scholar). Cystoviridae contains three dsRNA segments that are located inside a core particle composed of major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein (Nemecek et al., 2010Nemecek D. Heymann J.B. Qiao J. Mindich L. Steven A.C. Cryo-electron tomography of bacteriophage phi6 procapsids shows random occupancy of the binding sites for RNA polymerase and packaging NTPase.J. Struct. Biol. 2010; 171: 389-396Crossref PubMed Scopus (26) Google Scholar; Mantynen et al., 2018Mantynen S. Sundberg L.R. Poranen M.M. Recognition of six additional cystoviruses: Pseudomonas virus phi6 is no longer the sole species of the family Cystoviridae.Arch. Virol. 2018; 163: 1117-1124Crossref PubMed Scopus (25) Google Scholar, Mantynen et al., 2019Mantynen S. Sundberg L.R. Oksanen H.M. Poranen M.M. Half a century of research on membrane-containing bacteriophages: bringing new concepts to modern virology.Viruses. 2019; 11: 76Crossref Scopus (20) Google Scholar). The core particle is enclosed within a lipid-containing membrane. However, until now, there are only seven sequenced dsRNA phages yet. And six of them primarily infect Pseudomonas syringae, whereas phiYY is the only member that primarily infects P. aeruginosa with rough LPS (Yang et al., 2020Yang Y. Shen W. Zhong Q. Chen Q. He X. Baker J.L. Xiong K. Jin X. Wang J. Hu F. Le S. Development of a bacteriophage cocktail to constrain the emergence of phage-resistant Pseudomonas aeruginosa.Front. Microbiol. 2020; 11: 327Crossref PubMed Scopus (58) Google Scholar), which could be included into a phage cocktail to inhibit phage-resistant mutants during phage therapy and might be used to kill the rough LPS strains selected after chronic infection with antibiotic treatment (Hocquet et al., 2016Hocquet D. Petitjean M. Rohmer L. Valot B. Kulasekara H.D. Bedel E. Bertrand X. Plesiat P. Kohler T. Pantel A. et al.Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins.Environ. Microbiol. 2016; 18: 3482-3493Crossref PubMed Scopus (33) Google Scholar). P. aeruginosa is a common opportunistic pathogen that causes infections of bloodstream, urinary tract, burn wound, and airways of patients with cystic fibrosis (Waters and Grimwood, 2018Waters V. Grimwood K. Defining chronic Pseudomonas aeruginosa infection in cystic fibrosis.J. Cyst. Fibros. 2018; 17: 292-293Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Moreover, with the emergence of multidrug-resistant clinical isolates of P. aeruginosa, phage therapy has received renewed attention for treating multidrug-resistant bacterial infections (Smith et al., 2017Smith W.D. Bardin E. Cameron L. Edmondson C.L. Farrant K.V. Martin I. Murphy R.A. Soren O. Turnbull A.R. Wierre-Gore N. et al.Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis.FEMS Microbiol. Lett. 2017; 364: 9Crossref Scopus (71) Google Scholar; Waters et al., 2017Waters E.M. Neill D.R. Kaman B. Sahota J.S. Clokie M.R.J. Winstanley C. Kadioglu A. Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosa.Thorax. 2017; 72: 666-667Crossref PubMed Scopus (107) Google Scholar; Forti et al., 2018Forti F. Roach D.R. Cafora M. Pasini M.E. Horner D.S. Fiscarelli E.V. Rossitto M. Cariani L. Briani F. Debarbieux L. Ghisotti D. Design of a broad-range bacteriophage cocktail that reduces Pseudomonas aeruginosa biofilms and treats acute infections in two animal models.Antimicrob. Agents Chemother. 2018; 62 (e02573–17)Crossref PubMed Scopus (122) Google Scholar; Jault et al., 2018Jault P. Leclerc T. Jennes S. Pirnay J.P. Que Y.A. Resch G. Rousseau A.F. Ravat F. Carsin H. Le Floch R. et al.Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial.Lancet Infect. Dis. 2018; 19: 35-45Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar; Bao et al., 2020Bao J. Wu N. Zeng Y. Chen L. Li L. Yang L. Zhang Y. Guo M. Li L. Li J. et al.Non-active antibiotic and bacteriophage synergism to successfully treat recurrent urinary tract infection caused by extensively drug-resistant Klebsiella pneumoniae.Emerg. Microbes Infect. 2020; 9: 771-774Crossref PubMed Scopus (63) Google Scholar). Thus, a solid understanding of the phage-host interaction at the molecular level is valuable for the regulation and legislation of phage therapy in the near future (Rohde et al., 2018Rohde C. Resch G. Pirnay J.P. Blasdel B.G. Debarbieux L. Gelman D. Gorski A. Hazan R. Huys I. Kakabadze E. et al.Expert opinion on three phage therapy related topics: bacterial phage resistance, phage training and prophages in bacterial production strains.Viruses. 2018; 10: 178Crossref Scopus (79) Google Scholar). Transcriptomic approach is a powerful tool to study phage-host interactions and has been widely applied in studying phage-host interactions (Lavigne et al., 2013Lavigne R. Lecoutere E. Wagemans J. Cenens W. Aertsen A. Schoofs L. Landuyt B. Paeshuyse J. Scheer M. Schobert M. Ceyssens P.-J. A multifaceted study of Pseudomonas aeruginosa shutdown by virulent Podovirus LUZ19.mBio. 2013; 4 (e00061–13)Crossref PubMed Scopus (54) Google Scholar; Chevallereau et al., 2016Chevallereau A. Blasdel B.G. De Smet J. Monot M. Zimmermann M. Kogadeeva M. Sauer U. Jorth P. Whiteley M. Debarbieux L. Lavigne R. Next-generation -omics approaches reveal a massive alteration of host RNA metabolism during bacteriophage infection of Pseudomonas aeruginosa.PLoS Genet. 2016; 12: e100613Crossref Scopus (68) Google Scholar; De Smet et al., 2016De Smet J. Zimmermann M. Kogadeeva M. Ceyssens P.-J. Vermaelen W. Blasdel B. Jang H.B. Sauer U. Lavigne R. High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection.ISME J. 2016; 10: 1823-1835Crossref PubMed Scopus (73) Google Scholar; Zhao et al., 2017Zhao X. Shen M. Jiang X. Shen W. Zhong Q. Yang Y. Tan Y. Agnello M. He X. Hu F. Le S. Transcriptomic and metabolomics profiling of phage-host interactions between phage PaP1 and Pseudomonas aeruginosa.Front. Microbiol. 2017; 8: 548Crossref PubMed Scopus (24) Google Scholar; Li et al., 2020Li T. Zhang Y. Dong K. Kuo C.J. Li C. Zhu Y.Q. Qin J. Li Q.T. Chang Y.F. Guo X. Zhu Y. Isolation and characterization of the novel phage JD032 and global transcriptomic response during JD032 infection of Clostridioides difficile ribotype 078.mSystems. 2020; 5 (e00017–20)Crossref Scopus (15) Google Scholar; Lood et al., 2020Lood C. Danis-Wlodarczyk K. Blasdel B.G. Jang H.B. Vandenheuvel D. Briers Y. Noben J.P. van Noort V. Drulis-Kawa Z. Lavigne R. Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages.Environ. Microbiol. 2020; 22: 2165-2181Crossref PubMed Scopus (21) Google Scholar). It leads to a better understanding of the temporal transcriptional schemes, the impact of phage on host genes expression, and the regulatory mechanisms of phages. For example, the transcriptomic data of P. aeruginosa phage pap3 and its host revealed that phage early expressed protein Gp70.1 could affect host gene expression, and further experiment demonstrated that Gp70.1 binds to a global regulator Rpos to control host gene expression (Zhao et al., 2016Zhao X. Chen C. Jiang X. Shen W. Huang G. Le S. Lu S. Zou L. Ni Q. Li M. et al.Transcriptomic and metabolomic analysis revealed multifaceted effects of phage protein Gp70.1 on Pseudomonas aeruginosa.Front. Microbiol. 2016; 7: 1519Crossref PubMed Scopus (8) Google Scholar). Phages could also use phage encoded auxiliary metabolic genes to generate additional metabolites to support its replication (Zhao et al., 2017Zhao X. Shen M. Jiang X. Shen W. Zhong Q. Yang Y. Tan Y. Agnello M. He X. Hu F. Le S. Transcriptomic and metabolomics profiling of phage-host interactions between phage PaP1 and Pseudomonas aeruginosa.Front. Microbiol. 2017; 8: 548Crossref PubMed Scopus (24) Google Scholar). P. aeruginosa phage PAK_P3 significantly depletes bacterial transcripts to facilitate phage replication (Chevallereau et al., 2016Chevallereau A. Blasdel B.G. De Smet J. Monot M. Zimmermann M. Kogadeeva M. Sauer U. Jorth P. Whiteley M. Debarbieux L. Lavigne R. Next-generation -omics approaches reveal a massive alteration of host RNA metabolism during bacteriophage infection of Pseudomonas aeruginosa.PLoS Genet. 2016; 12: e100613Crossref Scopus (68) Google Scholar). Moreover, giant phage phiKZ could complete its infection cycle without the support from bacterial transcriptional machinery (Ceyssens et al., 2014Ceyssens P.-J. Minakhin L. Van den Bossche A. Yakunina M. Klimuk E. Blasdel B. De Smet J. Noben J.-P. Blaesi U. Severinov K. Lavigne R. Development of giant bacteriophage phi KZ is independent of the host transcription apparatus.J. Virol. 2014; 88: 10501-10510Crossref PubMed Scopus (93) Google Scholar), whereas another jumbo phage PA5oct relies on the host RNA polymerase to replicate (Lood et al., 2020Lood C. Danis-Wlodarczyk K. Blasdel B.G. Jang H.B. Vandenheuvel D. Briers Y. Noben J.P. van Noort V. Drulis-Kawa Z. Lavigne R. Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages.Environ. Microbiol. 2020; 22: 2165-2181Crossref PubMed Scopus (21) Google Scholar). In this study, we report the RNA sequencing (RNA-seq) analysis of dsRNA phage-P. aeruginosa interactions, reveal the gene expression patterns of both phage and host during infection cycle, and experimentally demonstrate that several deferentially expressed host genes are essential for phage life cycle. This study would contribute to the understanding of infection dynamics of dsRNA phage. Phage phiYY is effective in infecting the rough strains and has been included in a phage cocktail to limit the emergence of phage-resistance mutant (Yang et al., 2020Yang Y. Shen W. Zhong Q. Chen Q. He X. Baker J.L. Xiong K. Jin X. Wang J. Hu F. Le S. Development of a bacteriophage cocktail to constrain the emergence of phage-resistant Pseudomonas aeruginosa.Front. Microbiol. 2020; 11: 327Crossref PubMed Scopus (58) Google Scholar). PAO1r_8 is a phage-resistant mutant selected after dsDNA phage PaoP5 infection (Shen et al., 2018Shen M. Zhang H. Shen W. Zou Z. Lu S. Li G. He X. Agnello M. Shi W. Hu F. Le S. Pseudomonas aeruginosa MutL promotes large chromosomal deletions through non-homologous end joining to prevent bacteriophage predation.Nucleic Acids Res. 2018; 46: 4505-4514Crossref PubMed Scopus (26) Google Scholar). PAO1r_8 deleted 341 genes (PA1880-PA2220) and encoded 5,229 ORFs. It lost O-antigen because of the deletion of galU gene. It was renamed PAO1r in short in this study and selected as the host strain for RNA-seq analysis (Table S1). Next, a one-step growth curve of phiYY infecting PAO1r was measured (Figure 1A). The latency period is approximately 11 min, and the infection cycle duration is approximately 18 min, after which majority of the host are lysed. Thus, we focused on 6 (early), 12 (middle), and 18 min (late) time points after phage was added as representative snapshots of the phage infection cycle. The proportion of reads mapped as phage genes was fluctuating around 0.2%–1% (Figure 1B). This indicates that proportions of dsRNA phage transcripts are quite minor. On the contrary, the mRNA of dsDNA phage could reach a very high percentage. For example, the proportion of phage reads was over 80% and 60% for Acinetobacter baumannii phage phiAbp1 (Yang et al., 2019Yang Z. Yin S. Li G. Wang J. Huang G. Jiang B. You B. Gong Y. Zhang C. Luo X. et al.Global transcriptomic analysis of the interactions between phage phiAbp1 and extensively drug-resistant Acinetobacter baumannii.mSystems. 2019; 4 (e00068-19)Crossref Scopus (21) Google Scholar) and P. aeruginosa Podovirus LUZ19 (Lavigne et al., 2013Lavigne R. Lecoutere E. Wagemans J. Cenens W. Aertsen A. Schoofs L. Landuyt B. Paeshuyse J. Scheer M. Schobert M. Ceyssens P.-J. A multifaceted study of Pseudomonas aeruginosa shutdown by virulent Podovirus LUZ19.mBio. 2013; 4 (e00061–13)Crossref PubMed Scopus (54) Google Scholar), respectively. There are several explanations for the lower phage transcripts. First, dsRNA phages have a very small genome, for example, the sizes of the segments of phiYY were 3,004 (S), 3,862 (M), and 6,648 (L) bp (Yang et al., 2016Yang Y.H. Lu S.G. Shen W. Zhao X. Shen M.Y. Tan Y.L. Li G. Li M. Wang J. Hu F.Q. Le S. Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa.Sci. Rep. 2016; 9: 38795Crossref Scopus (34) Google Scholar). On the contrary, the genomes of dsDNA phages usually range from dozens to hundreds of kilo bases (Ceyssens and Lavigne, 2010Ceyssens P.-J. Lavigne R. Bacteriophages of Pseudomonas.Future Microbiol. 2010; 5: 1041-1055Crossref PubMed Scopus (75) Google Scholar; Shen et al., 2016Shen M.Y. Le S. Jin X.L. Li G. Tan Y.L. Li M. Zhao X. Shen W. Yang Y.H. Wang J. et al.Characterization and comparative genomic analyses of Pseudomonas aeruginosa phage PaoP5: new members assigned to PAK_P1-like viruses.Sci. Rep. 2016; 23: 34067Crossref Scopus (20) Google Scholar). Moreover, dsRNA phages encoded proteins to block bacterial transcriptional machines are not reported yet, whereas these proteins are common in dsDNA phages (Roucourt and Lavigne, 2009Roucourt B. Lavigne R. The role of interactions between phage and bacterial proteins within the infected cell: a diverse and puzzling interactome.Environ. Microbiol. 2009; 11: 2789-2805Crossref PubMed Scopus (85) Google Scholar). For example, bacteriophage Xp10 encoded protein P7 could bind to the host's polymerase to block host RNA transcription (You et al., 2019You L. Shi J. Shen L. Li L. Fang C. Yu C. Cheng W. Feng Y. Zhang Y. Structural basis for transcription antitermination at bacterial intrinsic terminator.Nat. Commun. 2019; 10: 3048Crossref PubMed Scopus (11) Google Scholar). The phage gene expression pattern can be clustered into early and late expressed genes (Figure 1C). Most genes from segment S and M are highly expressed at 6 min and decrease thereafter, whereas genes from segment L are highly expressed at 12 and 18 min. For phage phi6, RNA-dependent RNA polymerase within the phage particle transcribes S and M segments directly. However, the host protein YajQ is responsible for the activation of L transcription (Qiao et al., 2010bQiao X. Sun Y. Qiao J. Mindich L. Interaction of a host protein with core complexes of bacteriophage phi6 to control transcription.J. Virol. 2010; 84: 4821-4825Crossref PubMed Scopus (6) Google Scholar). Thus, we infer that segment S and M are immediately transcribed using RNA polymerase inside the phage particle and the initiation of L segment transcription might require a host protein(s), such as YajQ (PA4395), which results in delayed transcription of the L segment. The bacterial gene expression level was measured by FPKM (Reads Per Kilobase Per Million Read), and genes with log2 fold change values of 1.5 and q values of <0.05 were defined as differentially expressed genes (DEGs) (Table S2). Totals of 5.37% (281/5,229), 7.88% (412/5,229), and 8.89% (465/5,229) DEGs were identified at 6, 12, and 18 min after phage infection, respectively (Figure 2). The majority of the host genes (87.55%, 4,578/5,229) are not significantly affected by phage, indicating a modest impact of dsRNA phage phiYY on host gene expression, compared with dsDNA phages (Yang et al., 2019Yang Z. Yin S. Li G. Wang J. Huang G. Jiang B. You B. Gong Y. Zhang C. Luo X. et al.Global transcriptomic analysis of the interactions between phage phiAbp1 and extensively drug-resistant Acinetobacter baumannii.mSystems. 2019; 4 (e00068-19)Crossref Scopus (21) Google Scholar). RNA-seq data were validated by RT-qPCR. Fourteen DEGs were selected and the expression patterns were validated by RT-qPCR. The primers are listed in Table S3, and the RT-qPCR results are consistent with the RNA-seq results (Table 1).Table 1RT-qPCR ValidationHost GeneExpression Fold ChangesRNA-SeqRT-qPCR6 min12 min18 min6 min12 min18 minPA0499−7.235−4.878–−3.153−3.292–PA1176−9.204––−5.897––PA2849−2.968−3.453––PA3337−8.085−11.150−7.694−3.026−7.299−8.960PA1562–−3.315−3.669−3.225−3.095PA1864−10.768−11.763−25.108−3.205−6.056−11.834PA3879−5.178−6.564−7.127−4.221−6.887−6.211PA4238––3.010––2.322PA3609–10.84011.843–8.0878.310PA52394.1603.332–2.7452.303–PA47453.1513.009––PA28406.45495.9174.50311.1706.24917.238PA0140–55.40722.816–16.9868.389PA4613–81.668––142.15–Seven downregulated genes and seven upregulated genes of P. aeruginosa were selected from the RNA-seq dataset and validated by RT-qPCR. The 16S rRNA gene was used as the reference gene for normalization. The gene expression fold change in RT-qPCR was determined by the delta-delta Ct method. Open table in a new tab Seven downregulated genes and seven upregulated genes of P. aeruginosa were selected from the RNA-seq dataset and validated by RT-qPCR. The 16S rRNA gene was used as the reference gene for normalization. The gene expression fold change in RT-qPCR was determined by the delta-delta Ct method. According to the gene ontology (GO) analysis, the host DEGs can be clustered into several groups (Figure 3). Most of the upregulated genes are involved in the pathways of transcription and translation, because these resources might be essential for dsRNA phage replication. Most downregulated genes are clustered as chemotaxis, transcriptional regulators, adaptation and protection, amino acid metabolism, energy metabolism, and carbon compound catabolism. We wanted to test whether the DEGs are expressed passively or are functional for either supporting or blocking phiYY life cycle. Thus, we tested the effects of the six most upregulated and six most downregulated DEGs on phiYY infection, by making single insertional deletions of these genes in PAO1r and testing the efficiency of plating (EOP) of phiYY on each mutant. Among the 12 mutants, ΔPA4571, ΔPA3337 (ΔrfaD), and ΔPA0848 (ΔahpB) are completely resistant to phiYY, and the deletion of PA4613 (katB) decreased the EOP to 0.019 (Table 2).Table 2DEGs that Affect dsRNA Phage Infection EfficiencyStrainProtein Function (Gene)Expression Fold Change (log2)EOP (p Value)ΔPA4571Cytochrome c−3.09No plaqueΔPA5170Arginine/ornithine antiporter (arcD)−3.07(1)ΔPA2754Hypothetical protein−3.07(1)ΔPA0545Hypothetical protein−3.05(1)ΔPA22472-Oxoisovalerate dehydrogenase subunit alpha (bkdA1)−3.02(1)ΔPA3337ADP-L-glycero-D-mannoheptose-6-epimerase (rfaD)−3.02No plaqueΔPA0800Hypothetical protein5.4193.50 ± 1.07 (p < 0.05)ΔPA0140Alkyl hydroperoxide reductase (ahpF)5.793(1)ΔPA0849Thioredoxin reductase (trxB2)6.031(1)ΔPA4613Catalase(katB)6.3520.019 ± 0.03 (p < 0.05)ΔPA0848Alkyl hydroperoxide reductase (ahpB)7.065No plaqueΔPA3287Hypothetical protein7.131(1)The six most downregulated and upregulated host genes affected by phiYY were selected. Each gene was knocked out in PAO1r by insertional deletion. EOP of phiYY on each knockout strain was measured by comparing with that of PAO1r. (1): no significant difference of the EOP. Open table in a new tab The six most downregulated and upregulated host genes affected by phiYY were selected. Each gene was knocked out in PAO1r by insertional deletion. EOP of phiYY on each knockout strain was measured by comparing with that of PAO1r. (1): no significant difference of the EOP. Four of the six most upregulated genes are anti-oxidative stress genes, including alkyl hydroperoxide reductase (PA0848 and PA0140), catalase (PA4613), and thioredoxin reductase (PA0849). And ahpB and katB are important for phiYY infection (Table 2), which indicates that the host-encoded, anti-oxidative stress pathway is essential for dsRNA phage replication. This is reasonable, because RNA molecules are more sensitive to oxidative damages than DNA molecules. For DNA phages, the deletion of anti-oxidative genes had a modest impact on phage infectivity. For example, Campylobacter jejuni phage NCTC 12673 has an EOP of 0.15 on a katA mutant compared with that of a wild-type bacterium (Sacher et al., 2018Sacher J.C. Flint A. Butcher J. Blasdel B. Reynolds H.M. Lavigne R. Stintzi A. Szymanski C.M. Transcriptomic analysis of the Campylobacter jejuni response to T4-like phage NCTC 12673 infection.Viruses. 2018; 10: 332Crossref Scopus (32) Google Scholar). Thus, we infer that host-encoded anti-oxidative stress machines are essential to protect phage RNA genomes from oxidative damage and are essential for dsRNA phage infection cycles. Moreover, PA0800 is highly expressed during phage infection and the deletion of PA0800 increased the EOP to 3.5, indicating that this uncharacterized hypothetical protein might be involved in blocking phage infection. Although the function of PA0800 is unknown, its mechanism is a prospect for investigation in the near future. For the most downregulated genes, four of six of the genes did not affect phage cycle, whereas ΔPA4571 and ΔPA3337 completely resisted phage infection. PA3337 encodes an ADP-L-glycero-D-mannoheptose epimerase (rfaD), which is involved in the biosynthesis of the lipopolysaccharide precursor (Drazek et al., 1995Drazek E.S. Stein D.C. Deal C.D. A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression.J. Bacteriol. 1995; 177: 2321-2327Crossref PubMed Google Scholar). We performed adsorption assay to test the binding of phiYY to ΔPA3337 and PAO1r. The adsorption rate to PAO1r was ~90%, whereas it decreased to ~10% for ΔPA3337 (Figure S1). These data indicate that the deletion of PA3337 blocks phage adsorption and results in a complete loss of plaques. But its downregulation during phage infection is intriguing, as it may be unable to deter phage infection, seeing as the phage has already entered the bacteria. One possible explanation is to prevent further phage infection, after the first phage entering, which is similar to superinfection exclusion (Abedon, 2015Abedon S.T. Bacteriophage secondary infection.Virol. Sin. 2015; 30: 3-10Crossref PubMed Scopus (36) Google Scholar; Labrie et al., 2010Labrie S.J. Samson J.E. Moineau S. Bacteriophage resistance mechanisms.Nat. Rev. Microbiol. 2010; 8: 317-327Crossref PubMed Scopus (1420) Google Scholar). PA4571 is annotated as a cytochrome c oxidase, and the mechanism of PA4571 on phage infection is unknown. These data indicate that DEGs are not expressed passively but are the results of complicated bacterial defense mechanisms and dsRNA phage-encoded hijacking mechanisms. Some DEGs might be bacterial defense approaches to constrain phiYY infection, such as PA0800. On the contrary, some DEGs might be controlled by phages to assist phage infection cycles, such as the highly expressed anti-oxidative stress genes. Phages are viruses that are dependent on bacteria for replication. Thus, the host-encoded genes that are essential for dsRNA phage phi6, phi8, or phi2954 infection had been investigated previously. Mindich et al. have found that most of the cystoviridae family utilizes particular host proteins for the control of gene expression from the large genomic segment (Qiao et al., 2010bQiao X. Sun Y. Qiao J. Mindich L. Interaction of a host protein with core complexes of bacteriophage phi6 to control transcription.J. Virol. 2010; 84: 4821-4825Crossref PubMed Scopus (6) Google Scholar). Different dsRNA phages use YajQ, GrxC, GrxD, and Bcp for the induction of transcription of large L segments, which have guanine nucleotides missing at the first or second five prime positions relative to that found for segments S and M (Qiao et al., 2008Qiao X. Sun Y. Qiao J. Mindich L. The role of host protein YajQ in the temporal control of transcription in bacteriophage Phi6.Proc. Natl. Acad. Sci. U S A. 2008; 105: 15956-15960Crossref PubMed Scopus (20) Google Scholar, Qiao et al., 2010aQiao J. Qiao X. Sun Y. Mindich L. Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.Proc. Natl. Acad. Sci. U S A. 2010; 107: 6000-6004Crossref PubMed Scopus (9) Google Scholar). Moreover, by screening the transposon mutant library, they found mutations affecting pilus formation or LPS composition to be of consequence for susceptibility to infection of different dsRNA phages, because pilus or LPS is the phage receptor (Qiao et al., 2010cQiao X.Y. Sun Y. Qiao J. Di Sanzo F. Mindich L. Characterization of Phi 2954, a newly isolated bacteriophage containing three dsRNA genomic segments.BMC Microbiol. 2010; 19: 55Crossref Scopus (26) Google Scholar). However, in this study, we tested the effect of DEGs on phage infection cycle and found that some DEGs are essential for dsRNA phage infection, whereas PA0800 hinders phage infection. Since dsRNA phage phiYY only encodes 20 ORFs, it is reasonable to infer that dsRNA phage replication is massively dependent on host machinery. To predict the phage genes that might regulate host gene expression, the phage and bacterial gene coexpression analysis were conducted (Zhao et al., 2016Zhao X. Chen C. Jiang X. Shen W. Huang G. Le S. Lu S. Zou L. Ni Q. Li M. et al.Transcriptomic and metabolomic analysis revealed multifaceted effects of phage protein Gp70.1 on Pseudomonas aeruginosa.Front. Microbiol. 2016; 7: 1519Crossref PubMed Scopus (8) Google Scholar), which identified nine phage genes that have expression pattern correlations with host genes (Figure 4). The results indicate that phage genes, such as putative procapsid protein, RNA-dependent RNA polymerase, and packing NTPase, may play a central role in interacting with host genes. Many dsDNA phages encode proteins that could regulate the host gene expression (Roucourt and Lavigne, 2009Roucourt B. Lavigne R. The role of interactions between phage and bacterial proteins within the infected cell: a diverse and puzzling interactome.Environ. Microbiol. 2009; 11: 2789-2805Crossref PubMed Scopus (85) Google Scholar). For example, T4 phage uses Alc protein to cut off host transcription (Kashlev et al., 1993Kashlev M. Nudler E. Goldfarb A. White T. Kutter E. Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA.Cell. 1993; 75: 147-154Abstract Full Text PDF PubMed Scopus (39) Google Scholar). However, dsRNA phage phiYY has a limited genome with only 20 annotated genes, and most of these genes are involved in phage replication, packaging, attachment, and lysis (Yang et al., 2016Yang Y.H. Lu S.G. Shen W. Zhao X. Shen M.Y. Tan Y.L. Li G. Li M. Wang J. Hu F.Q. Le S. Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa.Sci. Rep. 2016; 9: 38795Crossref Scopus (34) Google Scholar), with eight genes that have yet to be characterized. To our knowledge, the impact of dsRNA phage-encoded proteins on host expression has yet to be studied. Thus, further studies of the phage genes might be interesting to test whether dsRNA phages could control and manipulate host gene expression and which phage protein(s) could upregulate the expression of anti-oxidative stress genes. This work represents the first RNA-seq analysis of the dsRNA phage infection processes, revealing dsRNA phage expression patterns and host responses. Furthermore, the phage-host interaction network analysis predicted some dsRNA phage-encoded proteins that may be involved in manipulating host gene expression. Finally, the host determinants of dsRNA phage susceptibility were investigated by studying the impact of significantly changed DEGs on phage infectivity, and the study reveals that phiYY is critically dependent on host machines to successfully replicate. These results raise new questions regarding host defense mechanisms against dsRNA phages and dsRNA phage-encoded hijacking mechanisms." @default.
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• W3048355891 date "2020-09-01" @default.
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• W3048355891 title "Transcriptomic Analysis Reveals the Dependency of Pseudomonas aeruginosa Genes for Double-Stranded RNA Bacteriophage phiYY Infection Cycle" @default.
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• W3048355891 doi "https://doi.org/10.1016/j.isci.2020.101437" @default.
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