| Pathogenic bacteria must have the ability to rapidly adapt to various environmental stresses during host infection,which is especially important for intracellular pathogens.Mycobacterium tuberculosis and Mycobacterium bovis are two typical intracellular pathogens that inevitably encounter various environmental stresses,such as hypoxia and nutrient deprivation,during host infection.It has been well established that the bacterial second messenger molecule cyclic di-GMP(c-di-GMP)plays a wide range of regulatory roles in a variety of important bacterial physiological processes and survival in adversity,often driving bacterial adaptation to various environmental stresses through specific transcription factor receptors.However,it is not clear whether c-di-GMP signaling molecules also play a regulatory function in intracellular bacteria such as Mycobacterium tuberculosis under hypoxic stress and the related regulatory pathways.In this study,using Mycobacterium bovis BCG strain as a model strain,we first identified and systematically resolved the novel mechanism by which c-di-GMP signaling molecules drive mycobacterial survival under hypoxic conditions through a novel receptor transcription factor Arg R.The specific results obtained are as follows.(1)Hypoxic stress significantly induced an increase in intracellular c-di-GMP content in M.bovis BCG strains,and the accumulation of c-di-GMP in the bacterial cytosol further promoted the ability of Mycobacterium to survive under hypoxic conditions.The intracellular c-di-GMP levels of the strains were measured under aerobic and hypoxic culture conditions using high performance liquid chromatography,and it was found that the intracellular c-di-GMP content of BCG bacteria increased 3.2-fold under hypoxic stress than under aerobic culture conditions.By comparing the growth differences of M.bovis BCG strains with different c-di-GMP levels measured under anoxic conditions,it was found that the survival numbers of bacteria with high c-di-GMP levels increased2.69-fold and 2.75-fold after 4 days and 6 days of hypoxia,respectively,compared to the control strains.Therefore,the accumulation of intracellular c-di-GMP in bacteria can significantly increase the survival of M.bovis BCG under hypoxic stress.(2)c-di-GMP positively regulates the expression of arginine synthesis gene cluster arg C-H and nitrate respiration-related gene cluster nar K2-X.A comparative analysis of gene expression differences between strains with high c-di-GMP levels and control strains under hypoxic conditions by bacterial transcriptome revealed that the expression of a total of 353 genes was significantly altered,with arginine synthesis gene cluster arg C-H and a previously reported nitrate respiration-related gene cluster nar K2-X associated with the hypoxic response of M.bovis being significantly upregulated in strains with high c-di-GMP level strains were significantly upregulated.Further overexpression of the arg C-H gene cluster in M.bovis BCG strains and assaying their survival under hypoxic stress revealed a2.32-fold increase in survival of the overexpressing strains compared to the control strains,suggesting that the arginine synthesis gene cluster arg C-H plays an important function in hypoxia adaptation in M.bovis BCG strains.(3)Arg R is a novel c-di-GMP receptor.c-di-GMP interacts with Arg R to relieve its inhibitory effect on arg C-H,resulting in increased intracellular arginine synthesis and decreased glutamate accumulation in bacteria.The arginine metabolism gene cluster arg C-H encodes multiple enzymes for the synthesis of L-arginine from L-glutamate,and Arg R acts as a repressor to negatively regulate the expression of the arg C-H gene cluster.By UV cross-linking and isothermal titration calorimetry(ITC)experiments,we confirmed that the transcription factor Arg R is a novel c-di-GMP receptor with a binding constant Kd of 0.341±0.05μM and a stoichiometric ratio of 1:1.By gel-blocking experiments,surface plasmon resonance,and semi-quantitative chromatin immunoprecipitation experiments,we found that c-di GMP showed significant inhibition of the DNA binding activity of Arg R both in vivo and in vitro.Further liquid chromatographic analysis of the arginine synthesis pathway revealed a 14.6%increase in the product arginine and a 17.9%decrease in the substrate L-glutamate in strains with high c-di-GMP levels.Thus,c-di-GMP significantly promoted the synthesis of arginine in bacterial cells,while reducing the accumulation of glutamate.(4)The transcription factor Arg R was induced to be expressed by c-di-GMP,and Arg R was able to interact with the transcription factor Cmr to enhance the regulation of nar K2-X by Cmr.c-di-GMP positively regulated the expression of arg C-H gene cluster and also promoted the expression of arg R.Bacterial two-hybrid and surface plasmon resonance experiments revealed that Arg R was able to physically interact directly with another transcription factor,Cmr.It was further revealed by gel-blocking experiments and surface plasmon resonance that Arg R interaction with Cmr promoted the binding of Cmr to the nar K2-X promoter,thus promoting the positive regulatory effect of Cmr,which ultimately enhanced bacterial intracellular nitrite transport and anaerobic respiration of bacteria.(5)c-di-GMP regulates mycobacterial adaptation to hypoxia by slowing bacterial aerobic respiration.L-glutamate is located at a critical site in the metabolism of Mycobacterium tuberculosis,and by measuring the intracellular ATP and NAD~+/NADH contents of BCG strains,it was found that the ATP content and NAD~+/NADH content in strains with high c-di-GMP levels were reduced by 51.8%and 73.2%,indicating that c-di-GMP inhibited aerobic respiration of bacteria.Further display of the oxygen content in the medium with methylene blue showed that oxygen was depleted in the control strain after24 h of anoxic treatment,while oxygen remained in the high c-di-GMP level strain,indicating that c-di-GMP slowed down the consumption of oxygen in the bacterial cells.In summary,this study identified a new pathway in Mycobacterium bovis by which the second messenger molecule c-di-GMP regulates bacterial adaptation to hypoxic stress through the Arg R receptor transcription factor:under hypoxic stress induction,the intracellular c-di-GMP content of Mycobacterium bovis increased significantly,which on the one hand relieved the Arg R repression of the arg C-H gene cluster and reduced bacterial intracellular glutamate accumulation.On the other hand,the Arg R protein,which was continuously induced by c-di-GMP,interacted with the Cmr protein to promote the positive regulation of the nitrate-reducing gene cluster nar K2-X by Cmr,thus facilitating the survival mode of anaerobic respiration in bacteria.These works expand our understanding of the diverse physiological functions of c-di-GMP and deepen our knowledge of the regulatory mechanisms of hypoxia adaptation in Mycobacterium tuberculosis,as well as provide potential new drug targets for blocking the infection process of Mycobacterium tuberculosis. |
