Pyrophosphatase LcpB Regulates Cell Wall Synthesis And Virulence In Staphylococcus Aureus

Staphylococcus aureus is a significant pathogen.It can cause skin and soft tissue infections,as well as fatal diseases such as sepsis,pneumonia,endocarditis and osteomyelitis.S.aureus is a classic gram-positive bacterium,and its cell wall is an important immunogenic substance and the target of antibiotics.Wall teichoic acid(WTA)is a unique component of the cell wall in gram-positive bacteria,but the specific mechanism of WTA synthesis has not yet been fully elucidated.LytR-CpsA-Psr(LCP)family is unique for gram-positive bacteria.Previous studies have shown that the LCP family may be involved in multiple physiological processes such as antibiotic resistance,virulence regulation,and membrane formation.To investigate the role of the LCP family in the synthesis of S.aureus cell walls,we used a clinical strain of methicillin-resistant S.aureus(MRSA)and selected LcpB as the research object.Results revealed that deletion of lcpB leads to the slow growth,increased autolysis,and irregular cell wall morphology in S.aureus.In order to investigate the mechanism of LcpB,we conducted RNA-seq,results combining RNAseq and RT-qPCR showed that lcpB deletion do not cause significant variation in the transcription level of genes related to cell wall synthesis.The enzyme activity test showed that LcpB has pyrophosphatase activity.There are six arginines in the extracellular segment of the LCP domain and they are the key enzyme activity sites of LcpB.Meanwhile,the synthesis of wall teichoic acid(WTA)in the lcpB knockout strain decreases,and the mutation of the key arginase activity site also leads to the reduction of WTA content in the cell wall.In order to investigate the role of LcpB in regulating the virulence of S.aureus,this study found that the lcpB mutant strain shows enhanced hemolysis,weakened ability to infect macrophage RAW264.7,and significantly enhanced virulence in mouse subcutaneous abscess model compared with wild type.Further study showed that the transcription level of virulence-related genes was significantly up-regulated in lcpB mutant strain,such as ehp,scnL,psma and psmfβ.In order to investigate the virulence regulation mechanism of LcpB,this study investigated the function of LcpB in N315 strain which has a deficiency in the Agr system naturally,and found that the effect of LcpB is independent of the Agr system.In addition,LCP proteins belong to cell wall stress stimulon,which will be activated when antibiotic pressure stimulation exists or cell wall synthesis-related enzymes are exhausted.This study confirmed that LCP family can respond to antibiotic stress through the two-component system VraSR.The results showed that the LCP family and VraSR specifically showed up-regulation of transcription with stimulation of the targeted cell wall antibiotics such as oxacillin,teicoplanin and vancomycin.However,the transcription level of LCP family will not be up-regulated in vraSR knockout strain even if there is antibiotic pressure.Electrophoretic mobility shift assay(EMSA)further showed that VraR could specifically bind to the promoter region of LCP proteins.In summary,this study identified the functions of LcpB,enriched the regulatory network of VraSR.This study has deepened our understanding of clinical CA-MRSA,and provided new potential drug targets for the development of anti-S.aureus drugs.