Study On Critical Gene PurC In Purine Biosynthesis Pathway Affects The Virulence Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae is an important animal pathogen that can cause porcine contagious pleuropneumonia with high morbidity and mortality.Due to the numerous serotypes of A.pleuropneumoniae and the increasing drug resistance,it is difficult to control and eradicate.The purine de novo biosynthesis,as one of the house-keeping pathways,is involved in the regulation of the virulence of pathogens.Blocking the de novo synthesis of purines could decrease the synthesis and secretion of virulence factors,iron uptake,signal transduction and other pathways,thereby affecting the virulence of pathogens.The intact purine de novo biosynthesis pathway plays a crucial role in the intracellular survival and virulence of various bacteria.However,the role of the purine de novo biosynthesis pathway in the regulation of A.pleuropneumoniae virulence has not been studied.In this study,the wild-type strain 4074 of Actinobacillus pleuropneum oniae was used as the parental strain to knock out the transcriptional regu lator gene purR and the key gene purC encoding phosphoribosylaminoimid azole-succinocarboxamide synthase,in the process of purine de novo biosy nthesis.The effect of the de novo synthesis of purines on the virulence of A.pleuropneumoniae is as follows:(1)PurR plays a negative regulatory role in the purine de novo biosynthesis in A.pleuropneumoniae.The deletion of purR gene increases the expression of genes related to purine de novo synthesis by 2-20 times,but it has no effect on the growth,virulence and stress tolerance of A.pleuropneumoniae.(2)The growth characteristic of the ΔpurC mutant in complete medium was not significantly different from the wild-type strain 4074,but in the chemical definition medium lacking adenine,the ΔpurC mutant had no ability to survive,indicating that the deletion of the purC gene blocked the purine de novo biosynthesis process.(3)In vitro simulated the high osmotic pressure and oxidative stress environment to explore the survival ability of ΔpurC mutant strain in adverse condition.The results showed that the osmotic and oxidative stress tolerance ofΔpurC was lower than that of the wild-type strain.(4)Mice were infected with ΔpurC gene mutant strain to explore the changes of the virulence.The mice in the ΔpurC-infected group were in good condition,with a survival rate of 83.33%,while all the wild-type and complementary strain-infected groups died,indicating that deletion of purC could significantly reduce the pathogenicity of A.pleuropneumoniae.In order to explore the proliferation ability in vivo,the blood and lungs of the mice were counted.At 24 h after infection,the viable count in the lungs of the mice in theΔpurC infection group was almost completely cleared and could reduce the pro-inflammatory factors after A.pleuropneumoniae infection at transcriptional level.(5)The cytotoxicity of ΔpurC to porcine alveolar macrophages(3D4/21)was significantly decreased and the clearance rate by 3D4/21 phagocytes was significantly accelerated.The adhesion of the ΔpurC mutant to neonatal pig tracheal epithelial cells(NPTr)was decreased.(6)The mice were immunized with ΔpurC as an attenuated vaccine,and the immune protection effect was evaluated.The results showed that the attenuated vaccine reduced the tissue damage caused by the wild-type strain,and the immune protection rate could reach 70%.In conclusion,deletion of the purC gene blocking the purine de novo biosynthesis of A.pleuropneumoniae.Mouse experiments and cytotoxicity experiments demonstrated that blocking the de novo synthesis of purines could reduce the virulence of A.pleuropneumoniae and alleviate the lung inflammation caused by A.pleuropneumoniae.Besides,deletion of purC could reduce the survival ability in adverse environment,cell adhesion and intracellular survival ability of A.pleuropneumoniae.Meanwhile,as an attenuated vaccine,ΔpurC can provide better immune protection effect against A.pleuropneumoniae infection of the same serotype,and provide a new target for the development of A.pleuropneumoniae vaccine.