| Actinobacillus pleuropneumoniae(APP)is the causative agent of Porcine Contagious Pleuropneumonia.The disease is mainly characterized by fibrous pneumonia and hemorrhagic pneumonia with high morbidity and mortality,and it has become a serious threat to the worldwide swine industry and the industry of hybridization wild boars.Stringent response is the self-control response of bacteria for the rapid perception and adaptation to environmental fluctuation,and is activated by a number of different starvation and stress signals.(p)ppGpp,the synthesis of which is catalyzed by RelA-a multidomain ATP:GTP(GDP)pyrophosphate transferase,is an important signal molecule during Stringent response.Therefore,the S8△relA strains wrere constructed by deletion of relA gene based on the A.pleuropneumoniae serotype 7(S8),using homologous recombination,kanamycin characters and sucrose counter-selection.Its characterization was studied preliminarily,laying a foundation for the further research of the adaptability and pathogenic in APP.The studies mainly include:Construction of deletion mutant S8△relA: according to the sequences of the relA gene(NC.009053.1)of serotype 7 APP S8 strain and pET28 a plasmid published in GenBank,primers were designed to amplify the up or down homologous arms of relA gene(relAS and relAX)and kan gene.These DNA fragments were connected by PCR then were cloned into the pEMOC2 vector,acquiring recomninant plasmid(pEMOC2-relAS-kan-relAX).Choose the E.coli β 2155 containing pEMOC2-rel AS-kan-relAX as the donor strain and the parental strain S8 as the recipient stain by conjugation and screen the single exchange strain which has Kanamycin and chloramphenicol resestance.After identification by PCR using primers P7/P8,the single exchange stains were picked to respectively copying on the Cm resistance plate and the 10 % sucrose and Kan resistant plate and screening CmS and KanR strains.After further identification by PCR,the kan gene fragment(816 bp)was amplified by P3/P4,and the relA gene fragment could not be amplified by P9/P10,so the clone could be identified as the deletion strain S8△relA.Characterization of deletion mutant S8 △ relA: S8 △ relA was continuously cultured in vitro and was identified by primers,indicating that the mutant has high genetic stability;Comparing with S8,the growth rate of S8 △ relA did not significantly change;After 24 h,during the stationary period of nutrient deficient environment,the viability S8 △ relA was decreased,showing a decline in its adaptability.By 20 different kinds of amino acids deficiency test,in the deficiency of the nutrient-Gly,Ile,Val,the growth ability was dramatically oppressed,suggesting that(p)ppGpp could sense the external pressure signalformed by the deficiency of Gly,Ile and Val.Persisters formation test: after 3 h 100 μg/mL florfenicol sterilization treatment,the proportion of living S8△relA with resistance phenotype was significantly decreased compared with S8,indicating the effect of(p)ppGpp on the formation of APP persisters.Simultaneously,the biofilm formation ability of S8 △ relA declined gradually with the prolongation of the culture time,and at 36 h the biofilm density is only 1/2 times of S8.In addition,the resistance to the phagocytosis by alveolar macrophages(PAM)was also significantly decreased in vitro.Animal experiment illustrated that the virulence of the S8△relA was declined by 2.7 times compared with the parental strain S8 in mice.In conclusion,these results demonstrated that the(p)ppGpp signaling regulated the adaptability and existed slight influence on virulence in A.pleuropneumoniae. |
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