Investigation On The Roles Of Actinobacillus Pleuropneumoniae YecSC In Sulfur Utilization And Infection

Actinobacillus pleuropneumoniae（APP）is a particularly anaerobic,gram-negative bacterium,and can cause pleuropneumonia in pigs.The disease has caused huge economic losses to the global pig industry.Recent studies have found that sulphur nutrient uptake and metabolic pathways affect the pathogenicity of pathogenic bacteria and are potential targets for pathogen therapy.Among these,cysteine/cystine is an important sulphur-containing amino acid and is associated with the virulence of some pathogenic bacteria.Studies have shown that E.coli Ydj N and YecSC-Fli Y are associated with cysteine/cystine transport,that E.coli Ydj N and YecSC-Fli Y homologous systems also exist in A.pleuropneumoniae,and that Ydj N and Fli Y are known to be involved in cysteine/cystine utilisation.However,the role of YecSC in transport,its interaction with Fli Y and Ydj N and its relationship with A.pleuropneumoniae pathogenicity need to be further investigated.Therefore,this study was carried out using A.pleuropneumoniae serotype 7 bacteria as material,and the specific experiments and results obtained are as follows:1.Construction and identification of A.pleuropneumoniae mutant strainsIn this experiment,a double-gene deletion mutant strain（35）yec SC was constructed by homologous recombination and negative screening of wild-type A.pleuropneumoniae serotype 7 strain（WT）.With the single-gene deletion mutant（35）ydj N previously constructed in the laboratory as the parent,the three-gene deletion mutant（35）yec SC（35）ydj N was constructed;The complementation mutant（35）yec SC（35）ydj N-yec SC⁺was obtained by electric transformation and resistance screening.2.Growth characteristics of A.pleuropneumoniae wild-type and mutant strainsTransfering WT,（35）yec SC,（35）yec SC（35）ydj N,（35）yec SC（35）ydj N-yec SC⁺to different media and measured OD₆₀₀ to analyze their growth ability.（35）yec SC（35）ydj N could not grow in chemically defined medium（CDM）with only cysteine or cystine as the sulfur source;The growth ability of the four strains in TSB and CDM medium containing other sulfur sources was basically the same.It shown that the deletion of yec S,yec C,and ydj N genes deletions could not use cysteine and cystine as sulfur sources3.Effect of YecSC and Ydj N deletion on the oxidative stress tolerance of A.pleuropneumoniaeWT,（35）yec SC,（35）yec SC（35）ydj N were exposed to PBS buffer containing 2.5 m M H₂O₂ for0.5 h and 1 h respectively,and calculated the survival rates of the three bacteria.We know that the survival rates of（35）yec SC did not reduced compared to WT,but the survival rates of（35）yec SC（35）ydj N were significantly lower than that of WT and（35）yec SC（P<0.01）,indicating that the YecSC and Ydj N play an important role in the oxidative stress defense mechanism of A.pleuropneumoniae.4.Determination of ATPase activityThe purified recombinant proteins r Yec S,r Yec C and r Fli Y were obtained by gene cloning,prokaryotic expression and affinity chromatography,and the ATPase activity of each recombinant protein was measured by peacock green colorimetric assay.The three proteins alone had no ATPase activity,while r Yec S and r Yec C together had some ATPase activity.The presence of r Fli Y could enhance enzymatic activities of r Yec S and r Yec C（P<0.01）,and in the presence of substrate cysteine,r Fli Y had significant enhancement effect on the enzyme activity（P<0.01）,while other non-related compounds did not.The results showed that the ATPase function of YecSC was dependent on the Fli Y protein and that the combination of Fli Y and specific substrates further enhanced the ATPase activity of YecSC;5.Effect of YecSC and Ydj N deletion on pathogenicity of A.pleuropneumoniae.To explore the impact of the blocked cysteine/cystine utilization and increased oxidative sensitivity caused by the YecSC and Ydj N deletion on the pathogenicity of A.pleuropneumoniae,mice were infected by intraperitoneally injection of WT,（35）yec SC,（35）yec SC（35）ydj N,and measured lung of colonized bacteria loads and competitive infection index.The result showed that there was no significant distinction of bacteria loads in the lungs and the competitive index of（35）yec SC vs WT and（35）yec SC（35）ydj N vs WT（0.77,0.57）was higher than the cutoff value（0.2）,showing that the deletion of yec SC and ydj N gene of A.pleuropneumoniae had no effect on the pathogenicity of mice.This study also determined the competitive infection index of two strains of（35）yec SC（35）ydj N and WT in the lungs of pigs,using the natural host of A.pleuropneumoniae,the pig,as a model.The results showed the competitive index of WT and（35）yec SC（35）ydj N in pig lungs was 1.38（（35）yec SC（35）ydj N vs WT）,which was higher than the cutoff value（0.2）,showing that the pathogenicity of A.pleuropneumoniae of ydj N and yec SC gene deletion on piglets did not decrease.In summary,this study confirms that the ATPase activity of A.pleuropneumoniae YecSC can be activated by Fli Y and specific substrates,that YecSC deletion affects Fli Y transport of cysteine/cystine,and that YecSC-mediated sulfur transport is associated with A.pleuropneumoniae resistance to oxidative stress but not with its pathogenicity.This study provides further insight into the mechanism of A.pleuropneumoniae sulfur nutrient transport and also provides a guide for subsequent studies on sulfur nutrient transport and metabolism and its association with A.pleuropneumoniae pathogenicity.