| Bacteriophage(phage)is a kind of virus that can infect bacteria.It has simple structure and samll size.They are the most abundant organisms and present in all ecosystems on Earth.Phage can specifically infect and kill bacteria and is a potential antibacterial factor that can be used to prevent and control bacterial infections,which is named Phage therapy.In addition to phage particles,phage-encoded products,such as lysozyme,have great application value in antibacterial infection.The phages that are most frequently discovered and studied are dsDNA phages with tails,while dsRNA phages are rarely reported.So far,more than 1900 phages have been completed for genome-wide sequencing,of which only six strains are dsRNA phages(phi6,phi8,phi12,phi13,phi2954 and phiNN),and they are all Cystoviridae.Most of their host is plant bacteria Pseudomonas syringae.Pseudomonas aeruginosa is a Gram-negative bacillus that is widely distributed in the environment.In clinical,this bacterium is a common conditional pathogen and it is also one of the important bacteria that causes nosocomial infections.Pseudomonas aeruginosa can resist antibiotic through intrinsic drug resistance and acquired drug resistance.Thus,it is particularly difficult for antibiotics to treat Pseudomonas aeruginosa infections,so in addition to antibiotic treatment,there is an urgent need to find new treatment methods.In this study,the clinical Pseudomonas aeruginosa strain,PA038,was used as the host,and the first dsRNA phage phiYY which can infect the Pseudomonas aeruginosa strain(PA038)was accidentally isolated from the sewage of Southwest Hospital.Genomic sequencing and comparative genomic analysis indicated that the phage belongs to the Cystoviridae.In the current study,the dsRNA phage phiYY in this study is the first Cystoviridae phage that can lyse Pseudomonas aeruginosa.It is of great significance to study the related information of phage phiYY.Therefore,this study analyzed the basic biological characteristics of phiYY and identified its host range.Then we expressed and purified the putative lysozyme in vitro that was encoded by phiYY.Subsequently,the activity and host range of the putative lysozyme have been identified.Finally,we designed a phage cocktail consisting of phiYY and other four different phages,and conducted a preliminary study on the strategy which about the formulation of phage cocktail.Furthermore,we proposed a new phage cocktail design strategy by experimentally determining the host spectrum of the cocktail and assessing its effects on the growth of the resistant strain.The main experimental contents and results of this study are as follows:1.Isolation and identification of a double-stranded RNA phage: The clinical Pseudomonas aeruginosa strain,PA038,was used as the host.And a phage which can infect PA038 was isolated from untreated sewage of Southwest Hospital.The chloroform test results and the electron microscopy results showed that the phage was a Cystoviridae phage.The phage genome was extracted and sequenced,which indicated this phage was a dsRNA phage of the Cystoviridae family.At present,only six Cystovirida phage strains have been studied,most of which were isolated from bacteria-infested legumes in the USA,and their hosts are Pseudomonas syringae.Then,the phage isolated in this study is the first member of Cystoviridae family which can infect the human pathogen Pseudomonas aeruginosa.Comparative genomics analysis and protein sequence alignment showed that lower similarity of the genome sequence was found between this phage and the other six Cystoviridae phages,but their protein sequences were similar.Phage evolution analysis showed that the phage has a certain homology with other six Cystoviridae phages,and has the closest affinity to phage phi13,so the phage isolated in this study is named phiYY.Finally,a panel of 233 isolated clinical Pseudomonas aeruginosa strains which were collected from seven different hospitals and the standard Pseudomonas aeruginosa strain PAO1 were selected as the test strains to identify the host range of phiYY.The results showed that phage phiYY can infect 99 isolated(about 40%)clinical Pseudomonas aeruginosa strains,indicating that it is a lytic phage with a broad lytic spectrum.2.A study on the activity of a lytic enzyme which encoded by dsRNA phage phiYY: Bioinformatics analysis indicated that the S fragment of phage phiYY genome contains a gene(orf17)encoding a putative lysozyme.The protein sequence analysis of the putative lysozyme showed that it contained two domains(lysozyme-like-family domain and the PG-binding domain),and its action site was predicted to be ?-1,4 glycosidic bond,which was existed in the peptidoglycan(PG)of cell wall.The lysozyme gene was recombined with the expression plasmid,and then transferred into Competent cell(BL21),finally a large amount of protein was induced to be expressed by IPTG and purified through Ni-NTA column in vitro.Enzyme activity experiments showed that the lysozyme could directly lyse Staphylococcus aureus and Pseudomonas aeruginosa whose outer membrane was damaged by the outer membrane permeabilizer,resulting in a significant reduction in the number of bacteria,but no direct killing effect was found on the Pseudomonas aeruginosa whose outer membrane was intact.The optimum conditions for the lysozyme are 37 °C,pH 7.5.In addition,a low concentration of EDTA combined with lysozyme can cause a synergistic effect.The bactericidal properties of lysozyme suggest that it can be used as a potential antibacterial agent.3.Study on a strategy for the formulation of phage cocktail: Phage cocktail is commonly used as a kind of therapeutic preparation in phage therapy,and its formulation is usually consisted with several phages with different host pectrums.If phage cocktail is widely used in clinic,it will inevitably lead to phage resistance,which is similar to antibiotic resistance.We considered optimizing the formulation of cocktail to make the cocktail has a broader host spectrum.In the meanwhile,it can kill the phage-resistant bacteria that maybe appeared in phage therapy.In the previous experiment,Pseudomonas aeruginosa standard strain PAO1 was used as the host,the brown mutant(PAO1r-1)and white mutant(PAO1w-1)that are resistant to phage PaoP5 were isolated after interaction with phage PaoP5.Sequencing revealed that PAO1r-1 and PAO1w-1 contained mutations in the genes galU and wzy respectively,which are the key genes involved in lipopolysaccharide(LPS)synthesis.So PAO1r-1 lost its O-antigen and PAO1w-1 has a truncated O-antigen.Therefore,this study intended to screen and isolate phages that can lyse PAO1r-1 and PAO1w-1 through different methods,then combine them into phage cocktail and further evaluate their lytic ability.In this study,we found that phiYY can effectively lyse PAO1r-1.The mutant phage PaoP5-m that can lyse PAO1w-1 was screened by co-cultivation of PAO1w-1 with PaoP5.In order to identify whether PaoP5-m is a PaoP5 mutant and its mutation site,the phage genome was sequenced.We found that its mutation site is A715 C of orf75.One mutant phage was randomly selected and named as PaoP5-m1 for further study.Subsequently,PaoP5,phiYY and PaoP5-m1 were combined with other two lytic phages(PaP1 and PaP8)into a phage cocktail for subsequent experiments.Results have shown that phage cocktail has a broader host spectrum and can effectively reduce biofilm formation than single phage,and can slow the growth of the resistant strains for a certain period of time.After co-cultivation of cocktail with PAO1 for a long time,six phage-resistant bacteria were randomly selected for genome-wide sequencing analysis.Two or more gene mutations were found in the genome of the phage-resistant bacteria.Theoretically,mutations in two genes required long-term evolution,suggesting that the cocktail formulation designed in this study may reduce the probability of phage-resistant bacteria production in a period of time.So,it maybe more suitable for clinical application than single phage for it can reduce the phage resistant that may occur in phage therapy.In summary,this study isolated a dsRNA phage phiYY which can lyse Pseudomonas aeruginosa,and identified its basic biological characteristics.The genetic information and characteristics were analyzed by genome sequencing,and comparative genomic analysis showed that it was closely related to the Cystoviridae phage phi13.Since phiYY belongs to the Cystoviridae family and has a broad host spectrum(about 40%,99 out 234),it is considered that the phage has great research and application value.In addition,this study expressed and purified phiYY-encoded putative lysozyme in vitro,which was confirmed to directly lyse Staphylococcus aureus and Pseudomonas aeruginosa with impaired outer membrane.Finally,we made a preliminary exploration about how to reduce the occurance of the phage resistant in phage therapy.A cocktail was generated by mixing phage phiYY with other four different host range lytic Pseudomonas aeruginosa phages showed a broader lytic spectrum than single phage and it may reduce the probability of emergence of phage-resistant bacteria in a period of time. |
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