The Regulatory Mechanism Of Metabolism And Infection By Signal Molecules In Gram-positive Bacterial Pathogens

Bacteria need different signaling regulatory system to adjust their metabolism pathways in response to changing environments.And bacterial pathogens strongly depend on effective signaling transduction system for host-bacterial interaction,escaping from host immune system and establishment of infection.The nucleotide second messengers and biotin are important bacterial signaling molecules,which regulate various bacterial physiological processes including central metabolism pathways,bacterial component synthesis,motility and virulence,etc..(1)The signaling pathways of c-di-AMP in Staphylococcus aureus small colony variantsS.aureus is very prevalent human opportunistic pathogen,which can cause serious infectious diseases in skin and airway.S.aureus small colony variants(S.aureus SCVs)are slow-growing,antibiotic-resistant mutants usually result from auxotrophism of menadione,hemin or thymidine.Thymidine-dependent SCVs(TD-SCVs)are associated with inactivation mutations of thymidylate synthase encoding gene thy A and their growth requires exogenous thymidine supply.TD-SCVs are shown to emerge after long-term trimethoprim sulphamethoxazole(SXT)treatment of patients with cystic fibrosis(CF),and are independently associated with more severe respiratory outcomes.c-di-AMP is and important nucleotide second messenger in bacteria but eukaryote lacks c-di-AMP.c-di-AMP is synthesized by diadenylate cyclases and degraded by its specific phosphodiesterases,involving in the regulation of central metabolism pathways,osmotic adaption and antibiotic resistant.It can also bind and active endoplasmic reticulum adaptor protein STING,resulting in type I immune response induction.In current study,a thymidine-dependent SCV and a hemin-dependent SCV were constructed by knocking out thymidylate synthase gene thy A and porphobilinogen synthase gene hem B in S.aureus Newman,respectively.?thy A induced higher STING-dependent inflammentory gene expression in both macrophages and murine lungs comparing to wild type and ?hem B.Both clinical TD-SCVs and ?thy A were found to produce higher levels of c-di-AMP by LC-MS/MS analysis.Bacterial intracellular c-di-AMP concentration was negatively correlated to exogenous thymidine levels,with lower thymidne leading to higher intracellular c-di-AMP levels.High c-di-AMP levels in ?thy A lead to hypermutability and induced higher inflammation upon infection.Our study provided a good explanation for why ?thy A is related to higher inflammation and worse lung outcomes.The PII family signal transduction protein Pst A was reported to be a c-di-AMP receptor with high specificity.In current study,bacterial two hybrid and in vitro enzyme activity assays revealed that Pst A interacted with thymidylate kinase TMK in a c-di-AMP dependent manner.Pst A/c-di-AMP complex promoted TMK activity and thymidine utilization upon binding,which would help ?thy A survive in environment with low thyminde levels.To our knowledge,it was the first time that c-di-AMP was reported to involve in bacterial central metabolism by regulating thymidine utilization.(2)The regulatory mechanism of c-di-GMP riboswitch in Bacillus thuringiensisB.thuringiensis is rod-shaped,gram-positive endospore forming insect pathogen.B.thuringiensis can produce parasporal crystal consisting of different insecticidal crystal proteins(ICPs)during the sporulation,which is broad spectrum pesticide.B.thuringiensis products are the most widely used microbial pesticide in the world.It is worthy to study the metabolism and cell cycle of B.thuringiensis.c-di-GMP is important bacterial second messenger,and regulates various physiological processes including virulence,cell cycle,biofilm formation and motility by binding to a wide variety of protein effectors and riboswitches.Its intracellular concentration is controlled through the antagonistic activities of diguanylate cyclases(DGCs)and c-diGMP-specific phosphodiesterases(PDEs).Riboswitches are regulatory RNAs typically located in the 5?-untranslated regions(5?-UTRs)of m RNAs to regulate the expression of downstream genes in response to changing concentrations of their cognate ligands.Canonical riboswitches consist of a conserved aptamer domain and a variable expression platform.In current study,a c-di-GMP riboswitch termed Bc2 RNA resides in the 5?-UTR of an m RNA that encodes collagen adhesion protein(Cap)in B.thuringiensis BMB171.In vitro transcription termination assays,as well as ?-galactosidase and quantitative PCR assays based on low and high c-di-GMP concentration mutants,indicated that the intrinsic transcription terminator in expression platform of Bc2 RNA strongly repressed the transcription of cap.Bc2 RNA performed a conformation change upon c-di-GMP binding and formed an anti-terminator,resulting in transcription of cap.Bc2 RNA deficiency lead to overexpression of cap,which then inhibited motility,exopolysaccharide secretion and biofilm formation,and at the same time promoted aggregation rates and affected virulence against its host insect Helicoverpa armigera.Our study revealed a new regulatory model for c-di-GMP riboswitch and named it as “repression/de-repression model”.Furthermore,Bc2 RNA was the the first experimentally validated c-di-GMP in Bacillus cereus group.(3)Biotin synthesis regulatory system mediated by transcription regulator Bio QTuberculosis caused by Mycobacterium tuberculosis infection is a chronic infectious disease,which generally affects the lungs,but can also affect other parts of the body.Most anti-tuberculosis drugs are only effective to active tuberculosis,but not to latent tuberculosis.With the emergence of multi-drug resistant strains of tuberculosis,studying mycobacterial pathogenesis and developing new anti-tuberculosis drugs is urgent.Biotin(vitamin H)is an essential micronutrient for three domains of life.Biotin acts as the co-factor of carboxylases in mycobacterium,so is critical for central metabolism and fatty acids synthesis in M.tuberculosis.M.tuberculosis lacks biotin uptake system,and depends on de novo biotin synthesis to maintain its normal growth.Deficiency of biotin synthesis related genes lead to growth and virulence defect.Given the fact that mammal lack biotin synthases,biotin synthesis pathway is a potential anti-tuberculosis drug target.Current research firstly studied the transcription profiles of biotin synthesis related genes using M.smegmatis as the model strain.Canonical biotin regulatory system depends on a bi-functional protein Bir A,which acts as both biotin-protein ligase(BPL)and transcriptional repressor for biotin biosynthesis pathway.In mycobacterium,Bir A lacks the function of transcriptional regulator.In current study,we indentified a Tet R family transcriptional regulator Bio Q,which compensated the fuction of mono-functional Bir A.We also identified the conserved DNA binding site(TGAAC-N3-GTTCA)of Bio Q by electrophoretic mobility shift and DNase footprinting assays.By knocking out bio Q,and associated with ?-galactosidase and quantitative PCR assays,we finally revealed the regulatory mechanism of Bio Q as a transcriptional repressor of biotin synthesis genes.