Polyphosphate Kinase Affects Oxidative Stress Response By Modulating CAMP Receptor Protein And RpoS Expression In Salmonella Typhimurium

After discovered in yeast in 1890 by L.Liberman,polyphosphate(polyP) was found in organisms from all three of life kingdoms.Despite inorganic polyphosphate's ubiquity-found in every cell in nature and likely conserved from prebiotic times-this polymer has been given scant attention.Among the reasons for this neglect of poly P have been the lack of sensitive,definitive,and facile analytical methods to assess its concentration in biological sources and the consequent lack of demonstrably important physiological functions.With the development of analyzing technology,research in recent years indicates that this ubiquitous biopolymer plays versatile and vital roles in response regulation and metabolism in prokaryotes and eukaryotes.Whereas the levels of poly P in mammalian sources are too low to be determined with confidence by previous methods,the enzymatically based assays identify and measure poly P unambiguously in all cells and tissues examined.Levels of 25-120 mM(in terms of Pi residues) in chains 50-800 residues long are found in rodent tissues(brain, heart,kidneys,liver,and lungs) and in subcellular fractions(nuclei,mitochondria, plasma membranes,and microsomes).It is reported that polyp can induce calcification in MC3T3-E1 cells and proposed that polyP,as a calcification inducer,can induce cell calcification is specific to osteoblast-like cells.It is also reported that polyp stimulates mammalian TOR,a kinase involved in the proliferation of mammary cancer cells.All of this suggested that polyp plays a role in cell differentiation and development -especially in bone tissues and brain.Besides,it is also reported that poly is present in platelet dense granules and is secreted upon platelet activation,a potent hemostatic regulator, serving to activate the contact pathway of blood clotting and accelerate factor V activation.In bacteria,polyP has found in relation to affect cellular structure,swimming,and products of extracellular polymers,polyP plays a negative role in the control of antibiotic production in Streptomyces lividans.As the enzyme responsible for synthesis of polyP,deletion of polyphosphate kinase(ppk) causes significant physiological changes.Alignment of ppk from different bacteria shows that ppk is highly conserved in many bacterial pathogens.It was demonstrated that ppk is essential for biofilm development,quorum sensing,and virulence of Pseudomonas aeruginosa.The ppk mutant of V.cholerae is significantly more sensitive to low pH, salinity,or H₂O₂ in minimal medium with low phosphate content.And it is presented that,in M.tuberculosis,downregulation of ppk lead to impaired its survival in macrophages and polyP plays a central role in the stress response of mycobacteria. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world.Despite its prevalence,relatively little is known about C.jejuni's precise pathogenesis mechanisms.It was reported that ppk is a pathogenesis determinant in Campylobacter jejuni.As to Salmonella Typhimurium and Gallinarum,inactivation of ppk differentially affects virulence and disrupts ATP homeostasis.In addition to the above specific functions,polyP/ppk was reported to participate in several fundamental cellular functions,such as DNA replicate,RNA degradation,protein degradation and so on.As an enteric pathogen,Salmonella Typhimurium is a typical Gram negative bacterium.It was reported that deletion of ppk in Salmonella Typhimurium and E.coli affects its transcription of rpoS,an important sigma factor for general stress response. We are interested in how ppk affects rpoS expression.In our study,we show that deletion of polyphosphate kinase(PPK),the principal enzyme responsible for synthesis of polyP,resulted in augmented expression of cAMP receptor protein(CRP) and rpoS and lowered H₂O₂ sensitivity in Salmonella Typhimurium ATCC14028.The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay,lacZ fusion,and exogenous cAMP addition,respectively,rpoS expression increased in cpdA(cAMP phosphodiesterase coding gene) mutant,further suggesting that cAMP-CRP upregulated rpoS expression.These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.