CydA Affects Plasma Agglutination By Regulating SaeRS In Staphylococcus Aureus

Staphylococcus aureus（S.aureus）distributes widely in the world and is one of the most common pathogenic bacteria in community-acquired and hospital infections.It can secrete a variety of exotoxins and enzymes,and coagulase is one of vital virulence factors.In the previous work,we found the ubiquinone cytochrome oxidase A subunit gene（cyd A）was related to plasma aggregation promotion by constructing a mutant library of S.aureus transposon.It was verified that the deletion of cyd A gene significantly reduced the ability to promote plasma agglutination.Ubiquinone cytochrome oxidase is a respiratory chain terminal oxidase in prokaryotes,which participates in NADH catabolism and provides power for ATP synthesis,and plays an important role in aerobic respiration of S.aureus.Objective1.To study the effect of cyd A on the energy metabolism in S.aureus.2.To explore mechanism of cyd A regulating the expression of plasma coagulase in S.aureus.Methods1.In normal gas and at 25,35 or 40 degrees as well as 6%CO₂ and 35 degrees,to detect the content of ATP and NAD+in the logarithmic growth phase of S.aureus Newman wild type strain and cyd A knockout strain.2.RNA sequencing was used to detect changes of genome expression of wild Newman strain and NewmanΔcyd A strain,exploring the effect of cyd A on gene expression of downstream genes,and q RT-PCR was used to verify the RNAseq results.3.To construct S.aureus Newman knockout strainΔsae RS and complementary strainΔsae RS::p RBsae RS by adopting homologous recombination techniques.4.Plasma agglutination test was used to observe the effect of sae RS knockout strain on the ability of promoting plasma agglutination.5.Considered that sae RS is a regulatory factor for downstream multiple virulence factors,as a result,hemolysis test and biofilm formation test were used to further verify sae RS function,to confirm whether cyd A affect plasma coagulase gene expression by regulating sae RS or not.Results1.The ATP content in NewmanΔcyd A strain has no significant change compared with Newman wild strain,while the NAD+content has decreased significantly under these different cultural conditions.2.The results of RNA-seq revealed that 136 genes were different expressed inΔcyd A strain compared with Newman wild type strain,including 48 were up-regulated genes and 88 were down-regulated genes.These genes are mainly enriched in these pathways,including infection,metabolism,synthesis as well as signaling pathways.3.Compared with the Newman wild type strain,the sae RS knockout strain failed to lead to plasma agglutination,and enhancement of plasma agglutination ability of genes overexpressed by Sae RS.4.The significantly reduced hemolytic ability and the improved biofilm formation ability was acquired after Sae RS and cyd A were knocked out.Conclusion:1.After Cyd A knockout,the content of NAD+in the intermediate product of energy metabolism of S.aureus decreased significantly,which suggested that Cyd A could affect plasma aggregation by reducing NAD+.2.After Cyd A knockout,the ability of bacteria to promote plasma agglutination decreased significantly.The regulatory mechanism is related to the decrease of gene expression of promoting plasma agglutination by down regulating SaeRS.