Physiological Function Of Econd Messenger Cyclic Diguanylate Signaling In Cobacterium Tuberculosis

People can be infected with Mycobacterium tuberculosis （MTB） by inhaling infected aerosols, and then MTB multiply in pulmonary alveoli and promote inflammation. Most people infected with MTB developed the typical granulomas due to fast duplication of MTB. Once the adaptive anti-MTB immunity is established, MTB stopped replicating and could persist in latent state for a long time. When induced by the biological and/or chemical agents, the latent MTB could resuscitate and proliferate, which was characterized as post-primary infection. The high rate of morbidity and drug resistance as well as the latency-resuscitation infection is the important mode of the infectious of MTB. The fact that China has the world’s second largest tuberculosis epidemic makes the study on MTB in details an urgent task.So far there are several known molecular mechanisms of the dormancy. Firstly, critical regulators for dormancy named dormancy survival regulator （dosR） can function through the regulation of the two-component system of dosR-dosS. Another major class of genes upregulated in persisters are toxin-antitoxin （TA） loci. Originally identified as plasmid maintenance factors, TA loci produce a toxin that is neutralized by a short-lived antitoxin. When daughter cells lose the genes carrying the TA loci, the antitoxins are degraded which allows the toxin to kill the bacteria. TA loci may become upregulated by environmental cues such as hypoxia and starvation, initiate a metabolic shut-down in the bacteria, and eventually facilitate dormancy. During evolution, MTB has developed diverse survival strategies in the phagosome to defend host stresses. The strategies include intracellular oxidative and nitrosative stress, detoxification of reactive oxygen intermediates and reactive nitrogen intermediates; protein repair and degradation; DNA repair, protection and mutagenesis and so on. So MTB become the typical intracellular pathogen, which display an important mechanism of bacterial chronic infection.Cyclic diguanylate （c-di-GMP） was first identified as an allosteric activator of cellulose synthase in the grape-associated bacterium Gluconacetobacter xylinus. The c-di-GMP biosynthetic and hydrolytic enzymes were identified as diguanylate cyclase （DGC） and phosphodiesterase （PDE） in Gluconacetobacter xylinus. DGC and PDE both contain two conserved domains, namely GGDEF and EAL. Phylogenetic distribution study suggested that GGDEF and EAL domains were widely present in bacteria, but not in archaea or eukarya. C-di-GMP can regulate various bacterial physiologic progresses including signaling communication between cells, biofilm formation, mobility, variation, virulence and so on. So far these results were obtained in fast growing bacteria. However, the mode of action of c-di-GMP in Mycobacterium tuberculosis （MTB） remained unclear. Can the c-di-GMP play an important role in MTB? Are there any small molecules or second messengers which can induce the dormancy of MTB in response to environmental changes?Our work has focused on elucidating the function of c-di-GMP during MTB infection. Firstly, we blasted in the genome of MTB H37Ra （attenuated strain） and H37Rv（virulent strain） with the bioinformatics methods. We found that there was a single GGDEF domain-containing gene, namely MRA₁362and Rv1354c, and a single EAL domain-containing gene, namely MRA₁365and Rv1357c respectively. The amino acid sequence is identical and the encoding product of MRA₁362can catalyze the GTP to c-di-GMP in vitro. Then, based on the mechanism of gene homologous recombination, we deleted the genes encoding the synthesize and degrading enzymes of c-di-GMP named MRA₁362and MRA₁365in MTB H37Ra, respectively. We found that△Ra1362formed biofilm more quickly than wild-type whereas△Ra1365formed biofilm more slowly than wild-type. The results of microarray showed that some genes related to dormancy were down-regulated in△Ra1362, which could be rescued by expressing the complementary plasmid and adding the exogenous c-di-GMP to the culture medium. We established the model of fast oxygen consumption and found that△Ra1362consumed oxygen faster than wild-type. But the level of intracellular NAD and NADH decreased in△Ra1362. We speculate that△Ra1362may compensate the non-sensing state by faster oxygen consumption. Based on these findings, we concluded that c-di-GMP in MTB regulates the biofilm development and the expression of some genes related to dosR by sensing the hypoxic and redoxic pressure. Rv1357c encodes the phosphodiesterase which degrades c-di-GMP in standard virulent MTB H37Rv strain. We deleted the Rv1357c gene and obtained△Rv1357c. After6weeks of infecting the C57BL/6mice, we found that the CFU in the lung of△Rv1357c group was almost the same as that of the wild-type group. However, the histopathologic was analysis showed difference. Comparing with the wild-type group, the solid lesions in△Rv1357c group were more obvious, and the weight of lung were bigger. These results suggested that the inflammatory reaction and the pathogenesis of the△Rv1357c group increased in infected mice. Our results demonstrate that MTB have difficulty in forming biofilm after sensing hypoxia due to increased bacterial proliferation and higher intracellular c-di-GMP level. The difficulty in forming biofilm will contribute to MTB spreading among tissues and result in more obvious inflammatory reaction. Our work showed for the first time that c-di-GMP can play a role as the second messenger in MTB by inhibiting the biofilm development in vitro and facilitating the pathogenesis in mice. C-di-GMP can also regulate the expression of some genes related to dormancy by sensing hypoxia.