| It is now widely accepted that bacteriophages are the most abundant microorganismson earth, which are10times more numerous than their hosts. Therefore, phages may playkey roles in bacterial evolution and population dynamics, and may have a great ecologicalimpact on the nature environment. Recently, with the help of next generation sequencing(NGS), immense populations of viruses are found in the human gut, oral cavity and otherbody sites. And the role of these phage populations in health and disease is beinginvestigated with great interest. However, in natural environments, bacteria are constantlythreatened by phage predation and therefore evolve to be more phage insensitive, while thephages have to subvert these processes to survive. Thus, phage resistance mechanisms mayhave key roles in regulating bacterial evolution and population dynamics in most naturalenvironments and human body sites. Bacteriophages and their bacterial host are constantlyengaged in co-evolutionary arm race. On the one hand, bacteria employ a wide range ofresistance mechanisms including prevention of phage adsorption and phage DNA entry,digestion of phage nucleic acids, abortive infection system and CRISPR/Cas system toevade phage infection or survive phage killing. One the other hand, bacteriophages arecapable of fast-evolved adaptive responses to hosts’ evolutionary change. Adsorption of thephage to bacterial host by a specifc interaction between phage proteins and receptors on thebacterial cell surface is the initial step of infection. Several receptors had been reported，including lipopolysaccharides (LPS), capsμLar polysaccharides (CPS), fagella and outermembrane proteins. As a defensive strategy, bacterial can modify these cell surfacereceptors, produce extracellμLar matrix or competitive inhibitors to prevent phageabsorption. Meanwhile, as a counter-defensive measure, phages are able to modify theirreceptor binding proteins, such as tail fiber, to achieve infection and kill the resistantbacterium. For example, Pseudomonas fluorescens SBW25was found to coevolve with itslytic phage phi2for more than300bacterial generations. This co-evolution is probably due to the continuous modification of the bacterial receptors and phage receptor bindingproteins.Pseudomonas aeruginosa is an important gram-negative opportunistic pathogen thatcauses serious infections in cystic fbrosis patients, cancer patients and otherimmune-compromised individuals. It remains one of the leading pathogens at most medicalcenters, partly due to its ability to develop resistance to conventional antimicrobials.Havinglong been proposed as a promising alternative to conventional antibiotics for treatingbacterial infection, bacteriophages are a group of viruses that are able to infect and killbacteria. In recent years, due to the growing drug resistance in a variety of pathogens andthe shortage of new antimicrobials, phage research, particμLarly bacteria-phage interaction,has regained the interest. Increasing effort has been devoted towards exploiting theapplication of phage-therapy in the treatment of bacterial infection. As an essential survivalstrategy of bacteria, phage resistance is one of the important aspects of bacterial-phageinteractions and also poses a serious obstacle for the application of phage-therapy.Therefore, a better understanding of phage-resistance mechanism is critical in revealing therelevance between phage ecology and bacterial ecosystem ecology, and is also crucial inhelping design successfμL phage-therapy strategies to treat/prevent bacterial diseases,including P. aeruginosa-related infections.We have recently isolated and sequenced three P. aeruginosa phages to explore theirbiological properties and interaction with host bacteria.Using one of the lytic phages (PaP1)and a clinical isolate ofP. aeruginosa strain (PA1), we studied their interactions:1. Mapping the tail fiber as the receptor binding protein responsible for differential hostspecificity of Pseudomonas aeruginosa bacteriophages PaP1and JG004. The first step inbacteriophage infection is recognition and binding to the host receptor, which is mediatedby the phage receptor binding protein (RBP). Recently, a single-particle trackingexperiment using fluorescently labeled phages accurately described the initial process. First,phages diffuse randomly until they encounter a bacterial cell. Then, the phages willcontinue to diffuse on the cell surface until they bind to a receptor that initiates the infectionprocess. Otherwise phages fall off from the cell surface and continue their free motion. Thebinding process is usually considered as two steps: reversible and irreversible binding. Forexample, in T4-like phages, the attachments of long tail fibers to specific receptors are reversible. After attachment via long fibers, baseplate changes its shape and six short fibersare generated and irreversibly binding to the LPS core. This process generates a signaltransmitted to the phage head and triggers DNA release. However, the baseplate ofTP901-1-like phages is in a conformation ready for host binding and thus do not rely onconformational change. The detailed mechanisms vary in different phages, but the y are alldetermined by both the receptor on the cell surface and the RBP in phage. So far,53Pseudomonas phages have been fully sequenced and submitted to NCBI, however, thereceptor-binding proteins of these phages have not been identified and the knowledge ofphage-host interactions is still very limited. This study aimed to identify and investigate thebinding specificity of the RBP of P. aeruginosa phages PaP1and JG004. These two phagesshare high DNA sequence homology but exhibit different host specificities. Thespontaneous mutant phage was isolated and exhibited broader host range compared with theparental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated asingle point mutation in ORF84(a putative tail fiber gene), which resμLted in thereplacement of a positively charged lysine (K) by an uncharged asparagine (N). We furtherdemonstrated that the replacement of the tail fiber gene (ORF69) of PaP1with thecorresponding gene from phage JG004resμLted in a recombinant phage that displayedaltered 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 mayhave potential value in phage therapy.2．Anovel phage resistance mechanism via chromosomal DNA deletion.Bacteriophagesare the most abundant organisms on earth, and are estimated to be tenfold more than theirbacterial hosts. In order to survive phage predation, bacteria develop a broad range of phageresistance mechanisms, such as preventing phage adsorption, digesting phage nucleic acids,abortive infection and CRISPR/Cas system.In this study, Pseudomonas aeruginosa PA1strain was infected with lytic phage PaP1and PA1strain exhibited high frequency (10-5) indeveloping phage-resistant mutants. Intriguingly, a high percentage (~30%) of thesemutants displayed red pigmentation phenotype (Red mutant). Via comparative genomicanalysis, one Red mutant PA1r was found to have a219.6kb genomic fragment deletion,which contains two key genes hmgA and galU. Deletion of hmgA resulted in theaccumulation of a red compound homogentisic acid, the substrate of hmgA; while a galU mutant is devoid of O-antigen which is required for phage adsorption. Further analysisrevealed the deletion of the genomic fragment containing galU and hmgA gene could bedetected in all other Red mutants, and thisnew deletion events exhibit three clearfeatures:large genomic fragment deletions (200~300kb), ahigh frequency (10-6), and nosite-specificity.In summary, this study suggested a novel phage resistance pathway bychromosomal DNA deletion. |
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