Investigation On The Function And Pathogenicity Of Genes Related With Sulfur Transport In Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP)is a Gram-negative pathogen that can cause porcine pleuropneumonia.The high infectivity and lethality of swine pleural pneumonia has caused huge losses to the economy of China even all over the world.Sulfur is a vital nutrient for bacteria due to its critical role in cells as a component of many fundamental organic molecules,Moreover,sulfur is involved in several cellular processes,including energy transduction,redox homeostasis,In addition,sulfur metabolic pathways are essential for the survival and expression of virulence of many pathogenic bacteria.As an invasive bacterium,A.pleuropneumoniae appears to be affected by host nutritional immunity,and it is accepted that its virulence is related to nutrient utilization system(s).however,knowledge of sulfur acquisition in this bacterium is poorly understood.Consequently,to further clarify the pathogenesis of A.pleuropneumoniae,there is a need to further investigate the molecules responsible for sulfur utilization.The process and main conclusions are summarized as follows:1.Analysis of the growth characteristic of sbp mutant in vitroThe sbp mutant ?sbp was constructed based on A.pleuropneumoniae WF83,and the complemention mutant C-?sbp was constructed based on ?sbp by electrical transformation.To assess the role of Sbp on bacterial growth and the utilization of sulfur sources,the growth curves were drawn by inoculating WF83,?sbp and C-?sbp in TSB and different CDM formulas.The results suggested that Sbp is involved in bacterial growth and plays an important nutritional role for A.pleuropneumoniae in relation to potassium sulfate and methionine utilization.2.Analysis of the effect of Sbp on the Virulence of A.pleuropneumoniae.To evaluate the role of Sbp in the virulence of A.pleuropneumoniae,The 50%lethal dose(LDso)of A.pleuropneumoniae WF83,?sbp and C-?sbp was determined,a mouse survival and mortality study and the bacterial in vivo colonization assay was also carried out.Besides,a pig infection assay was performed,and in vivo competitive index(CI)were determined.The results indicated that there were no difference in the LDso,the same as in the amount of bacteria in the lung tissue of mice,the CI in pigs was much higher than the weakening threshold,indicating that Sbp had no effect on the pathogenicity of A.pleuropneumoniae.3.Analysis of the function of FliY and YdjNThe ?fliY,?ydjN and ?fliY?ydjN were constructed based on A.pleuropneumoniae WF83.To investigate the role of FliY and YdjN on bacterial growth and the utilization of sulfur sources,the growth curves were drawn as previously described.To evaluate the role of FliY and YdjN in the virulence of A.pleuropneumoniae,the bacterial in vivo colonization assay and a mouse infection assay was performed,and in vivo competitive index(CI)were determined.The results indicted that FliY and YdjN is not involved in bacterial growth in TSB;However,The CDM showed that A.pleuropneumoniae lost the ability to utilize cystine and cysteine after double deletion FliY and YdjN,the thiamine is not neseccery while thiamine can significantly promote the utilization of cysteine and cystine;There was significant difference in Carrying capacity in lung tissue of mice,the CI was 0.22,and was higher than the weakening threshold(0.2),and indicated double deleted FliY and YdjN might reduce the pathogenicity of A.pleuropneumoniae,and the uptake of cystine and cysteine may be the pathogenic factor of A.pleuropneumoniae.Through this study,the characteristics of Sbp,FliY and YdjN in A.pleuropneumoniae sulfur utilization and effects on bacterial pathogenicity were preliminarily determined.Sulfur is an essential nutrient for A.pleuropneumoniae.A.pleuropneumoniae can obtain sulfur nutrition in a variety of ways:Sbp is responsible for the transfer of sulfate and methionine,and FliY and YdjN are responsible for the uptake of cystine and cysteine in a synergistic manner.Although Sbp has nothing to do with the pathogenicity of A.pleuropneumoniae,the double deletion of FliY and YdjN can reduce the pathogenicity of A.pleuropneumoniae in mice.It can be seen that the acquisition of sulfur nutrition(cystine and cysteine)is likely to be one of the key factors of A.pleuropneumoniae's pathogenicity.This study provides a theoretical basis for further research on the sulfur metabolism of A.pleuropneumoniae and the control of porcine pleurisy.