Characterization Of Environmental Stress Response By CRP,a Novel Expression Regulatory System Of Staphylococcus Aureus

Staphylococcus aureus is one of the main pathogenic bacteria causing digestive and mammary gland diseases in livestock and poultry,which severely restricts the healthy development of animal husbandry.Antibiotics are the main means to kill Staphylococcus aureus,but with the introduction of China’s feed ban,there is an urgent need to develop new methods to prevent and control Staphylococcus aureus infection.In the process of S.aureus infection,environmental stresses such as acid,salt,antibiotics,hypoxia and oxidative stress can affect its survival and reproduction ability.Meanwhile,S.aureus can be expressed by a complex network of regulatory genes,regulating the expression of various virulence factors,resistance factors and immune escape factor protein,resistant to environmental stress.Therefore,the systematic study of the tolerance role of Staphylococcus aureus to environmental stress and related mechanisms is of significance for the prevention and control of jokes and mammary system diseases caused by Staphylococcus aureus in livestock and poultry.Global transcription factors are the core factors for bacteria to regulate downstream functional gene expression and respond to environmental stress.So far,researchers have discovered more than 20 regulatory systems in Staphylococcus aureus,but the functions and regulatory mechanisms of more than 50%of the genes in Staphylococcus aureus are still unknown.c AMP receptor protein is a global transcription factor ubiquitous in various prokaryotes such as Escherichia coli,Salmonella,Klebsiella pneumoniae and Streptomyces.It can respond to many environmental signals comprising c AMP,NO,CO,hypoxia,redox state,oxidative stress,temperature stress and the like nitriding.But S.aureus in the CRP system has not been reported.Therefore,we conducted in-depth research on the discovery and function of the Staphylococcus aureus CRP system,and obtained the following research results:In the previous study,our laboratory discovered a transcription factor X protein with unknown function,which can specifically bind to the promoter of the Staphylococcus aureus recombinase ccr C2 gene.In this paper,the Motif analysis of X protein found that its N-terminal has a c NMP binding site,C-terminal has a DNA binding site,which is presumed c AMP receptor protein CRP.E.coli prokaryotic expression strain BL21（DE3）was used to express and purify the 6×His-taged X protein,and a polyclonal antibody of X protein was prepared.Affinity chromatography and Western blot experiments proved that the X protein has a specific binding capacity to biotin-labeled c AMP,and the negative control protein Sig B has no affinity,which proves that the X protein is a CRP protein.In order to detect whether the CRP in S.aureus can respond to various environmental stresses,we use different environmental factors to treat S.aureus strain BA01611,and then detect the difference in expression of crp gene by q RT-PCR test.The results showed that treatments such as addition of Zn²⁺,addition of Cu²⁺,lack of Fe³⁺,addition of glucose,and lack of oxygen significantly down-regulated the expression of crp,sub-inhibitory concentration of the antibiotic raised crp expression,and high salt environment of the crp no significant expression.These results indicate that the CRP protein in Staphylococcus aureus can respond positively to environmental stress.To clarify the relationship between CRP and Staphylococcus aureus phenotype,this paper successfully constructed crp gene knockout strains BA01611-△crp,Mu50-△crp and Newman-△crp through homologous recombination,and detects the biological phenotype of each strain.The results show:compared with the wild-type strain,the staphyloxanthin production capacity of Mu50-△crp and Newman-△crp is weakened,and the staphyloxanthin production capacity of BA01611-△crp has no significant change;BA01611-△crp and Mu50-△crp have enhanced resistance to oxacillin,but Newman-△crp has no significant change in its resistance;The hemolytic ability of BA01611-△crp and Mu50-△crp was enhanced,but the hemolytic ability of Newman-△crp had no significant change;The biofilm formation capacity of Newman-△crp was enhanced,while the biofilm formation ability of BA01611-△crp and Mu50-△crp did not change significantly..The staphyloxanthin is a key factor that determines the pathogenicity of S.aureus.It can help S.aureus resist the damage of active oxygen in host neutrophils and improve the anti-oxidation ability of S.aureus.In this paper,q RT-PCR was used to detect the expression of various genes in the staphyloxanthin synthesis gene cluster crt OPQMN and the regulatory operon rsb UVWB in Mu50 and Mu50-△crp.It is speculated that CRP may stimulate the activity of Sig B by promoting the expression of rsb U,thereby enhancing the biosynthesis of S.aureus pigment.Combined with the existing regulatory network information,it is speculated that CRP may stimulate the activity of Sig B by promoting the expression of rsb U,thereby enhancing the staphyloxanthin biosynthesis of S.aureus.In summary,this experiment verified the presence of the c AMP receptor protein CRP in S.aureus.It is found that multiple environmental factors can affect the expression of crp.CRP has the function of promoting staphyloxanthin production of S.aureus,reducing its resistance toβ-lactam antibiotics,decreasing its hemolytic capacity,and reducing its biofilm production capacity.CRP may activate the activity of Sig B by promoting the expression of rsb U,which in turn enhances the biosynthesis of staphyloxanthin.The above study provides a theoretical basis for regulating the expression of the transcription factor CRP and thus inhibiting the pathogenic ability of S.aureus and reducing the risk of healthy breeding by S.aureus infection in animal husbandry.