Study On Biological Characters Of S. Paratyphi A Phage LSPA1 And Interations With Host

Salmonella paratyphi A is the cause of paratyphoid pathogen, which is spread by the fecal-oral route. Phage is bacteria viruses, which can infect and lyse host cells. Phage prevalently exists in the host bacteria, in most conditions, phage and host bacteria are involved in ongoing joint evolution, and reach the host-phage homeostasis in nature. Currently, the mechanism of phage and host interaction is not clear. This study screened a potent phage from S. Paratyphi A (LSPA1) and its biological characterristics and genomic were analyzed. Moreover, the interactions of LSPA1 and S.Para typhi A were studied. And thus a deeper under- standing of the phage and the interactions between phage and host were gained.Presently, treatment of pathogen infection is mainly using antibiotics. However, abuse of antibiotics and biofilm formation greatly reduces the effect of antibiotic treatment, so it is particularly important to find a novel method. Phage, with strong infectivity and host specificity, which is a deadly threat for host and thus researchers envisioned to cure pathogen infection by phage therapy.In order to obtain a new phage, after mixed and cultured the hospital sewage and S. Paratyphi A and the S.Paratyphi A phage (LSPA1) was separated and purified. The biological characteristics of LSPA1, including best Multiplicity of infection (MOI), pH and temperature sensitivity, structure proteins and initial receptors. LSPA1 genome was extracted and sequenced by shotgun. The evolutionary tree, homology and open reading frames were characterized. To study LSPA1 and S. Paratyphi A interactions, at first anti-mutant phage strains of S. Paratyphi A was screened by using combination transposition, and then the inserted genes were identified by asymmetric PCR and T-A cloning. And site-directed mutagenesis of homologous recombination and complementation tests further proved that due to mutations in these genes made mutant strain of S. paratyphi resist to phage. Moreover, the mutation of σ-54-dependent translation regulator gene was associated with biofilm formation.Phage screening results show that:1) the hospital sewage and S. Paratyphi A mixed culture and centrifuged, the supernatant was used to screen phage. Transparent plaques appeared on the double flat, which indicated lytic phage from S. Paratyphi A was successfully screened in the hospital sewage, named LSPA1.2) The phage LSPA1 was concentrated to observe in electron microscope, the results showed that the phage LSPA1 has a 64-sided head and a long tail suggesting that LSPA1 belongs to caudata virus.3) Extraction of the phage genome and restriction with enzyme digestion, the results show that the genomic can be digested and proved that phage LSPA1 is double-stranded DNA virus.Analysis of phage LSPA1 biological characteristics:1) Inoculated with different MOI phages infect host, the results show with an MOI of 0.2 gained the highest phage titers proved that the best MOI of LSPA1 is 0.2.2)The growth curve of phage LSPA1 demonstrates that the incubation period is 20 min, the outbreak period is 50 min and the amount of outbreaks are 129.3) After phage LSPA1 treatment with different temperatures and pH its titer were measured. The result illustrated that bacteriophage LSPA1 has a high titer at pH between 3 and 10 and pH among 1 to 2 or 11 to 12 the phage lost their activities. What’s more, with increasing temperature, the titers significantly decreased and at 80℃, almost no activity phage exists.4) SDS-PAGE analysis of phage LSPA1 show that mainly contains four proteins which their relative molecular are about 67KD,60KD,55KD,40KD, respectively. The 55KD protein belongs to capsid proteins.5) The phage receptors, lipopolysaccharide and outer membrane proteins were preliminarily analyzed.Phage LSPA1 genomics analysis:1) full length genomic of phage LSPA1 are 41880 bases which base composition are A=25.09%, T=24.43%, G=25.13%, C= 25.35%, G+C= 50.48%.2) phage LSPA1 genome contains 58 ORF,38 of which are in positive strand,20 of which in the negative strand.3) LSPA1 not only have a high affinity with Salmonella phages, and also has a high affinity with E.coli phage, and has highly similar with SETP3 genomes.4) The complete genome sequence of bacteriophage LSPA1 was submitted to GenBank, and its accession number is KM272358.Phage LSPA1 and host S. Paratyphi A interactions:1) six anti-phage mutants were screened, and proteins encoded by these mutation genes are lysozyme, hypothetical protein (probably methyltransferase), thiosulfate reduction electronic precursor enzyme, assuming the sensor kinase protein, acetolactate synthase large subunit III isoenzyme, assuming σ-54-dependent translation regulator.2) the biofilm shape of assumed a-54-dependent translation regulator mutant was significantly changed and the ability of forming biofilm was significantly enhanced.3) site-directed mutagenesis and complementation experiments further demonstrated assume a-54-dependent translational regulation is anti-phage and biofilm-related genes, and σ-54-dependent translation regulator missing host exhibited different characteristics, such as anti-phage, biofilm forming significantly enhanced.In this study, a lytic phage strain of S. Paratyphi A from the hospital’s sewage was screened and its biological properties were analyze, which proved that LSPAl belongs to a double-stranded, long-tailed phage. Analysis of LSPA1 genomics and interaction with host S. Paratyphi A, which demonstrated six genes were associated with phage infection. Furthermore, a-54-dependent translation regulator deletion can significantly enhance the ability of biofilm forming. This study will help further study the mechanism of phage infection and consequently contribute to therapy pathogen infection by phage.