Cyclic Di-GMP Signaling Regulates Mycobacterium Tuberculosis Dormancy, Biofilm Development And Virulence

Tuberculosis is a bacterial contagious disease, majorly caused by Mycobacterium tuberculosis （MTB）, which kills about2million people worldwide each year. MTB has excellent dormancy ability, resulting in latent infection of one third of the world’s population and long medication cycle of more than half a year. The mechanisms underlying MTB dormancy need extensive and thorough investigations, although it has recently been demonstrated that a three-component regulatory system, DosR/DosS/DosT, is part of a programmed strategy employed by the bacilli to survive from dormancy. It is anticipated that discoveries of the dormancy mechanisms will pave a way for development of novel anti-tuberculosis vaccines and drugs, and are thus vitally important for controlling tuberculosis epidemic and shortening cure period.Cyclic di-GMP （c-di-GMP） is an intracellular second messenger only found in bacteria, which regulates various important bacterial social behaviours such as virulence, biofilm development and motility. However, the physiological function of c-di-GMP and its regulatory mechanism in MTB remain unknown.We predicted by bioinformatics that MTB genome encodes each one gene of c-di-GMP synthesizing and degrading enzymes which are conserved in all MTB strains with the accession numbers of Rv1354c and Rv1357c in the virulent strain H37Rv and MRA₁362and MRA₁365in the avirulent strain H37Ra, respectively. Rv1354c is consisted of the three domains of GAF, GGDEF and EAL, while Rv1357c only habours an EAL domain. Rv1354c, heterologously expressed in Escherichia coli as a soluble active form, is a dimer and can transforms GTP and GDP into c-di-GMP. Interestingly, Rv1354c shew higher activity under a reduced state than a oxidized condition.The gene knockout mutants of MRA₁362or MRA₁365without any heterologous marker, named as△Ra1362and△Ra1365respectively, were obtained by a multi-step screening after transforming their target knockout plasmids into H37Ra. The corresponding gene-complemented strains, Com-Ra1362and Com-Ra1365, were also constructed. No obvious difference in growth was observed between the four strains and the wide-type （WT） H37Ra.Total RNA samples of each MTB culture at late-growth state were subjected to microarray analysis at a whole transcript level. Many genes were differentiatedly expressed in ARa1362or ARa1365compared to WT H37Ra and Com-Ra1362/Com-Ra1365. Interestingly, approximately half of the significantly differentially-expressed genes belongs to the known dormancy genes of the DosR regulon. The expression defects for these genes in ARa1362could be recovered in Com-Ra1362or by addition of synthetic c-di-GMP into the ARa1362culture. In contrary to ARa1362, several dormancy genes were up-regulated in ARa1365. The down-regulation of a part of dormancy genes in ARa1362was futher verified by quantitative RT-PCR by using the cultures at OD6oo=1.6for analysis.ARa1362has the strongest ability of adherent bio film formation, followed by WT, and ARa1365is the weakest one. In consistance with this, the biofilm development of ARa1362at air-liquid interface was the fastest:a typical bacterial biofilm structure with rich wrinkles was observed for ARa1362at a21-day culture when only a thin cell layer of WT H37Ra or ARa1365was floating on the liquid surface; however, there was no obvious difference in the biofilm shapes after5-week growth for the three strains.An in vitro anaerobic model was used to compare the dormancy ability of ARa1362with WT H37Ra. ARa1362used up oxygen at160h, faster than WT H37Ra（172h）. The intracellular NADH levels were nearly identical for them during the whole culture process, whereas the NAD level and thus the NAD/NADH ratio in ARa1362were lower than those in WT H37Ra when they entered into hypoxia at6d. In accordance with the balance disturbance, ARa1362lost90%of its viability in comparison with WT H37Ra when their8to10-day cultures were allowed to grow on plates. Furthermore, ARal362need more of5-7d to form visible colonies than the latter. The data gained by quantitative RT-PCR indicated that several dormancy genes was down-regulated in ARa1362at an early anaerobic state although the dosR mRNA level in ARa1362was slighly higher than that in WT H37Ra.Rv0195, one of the targets of c-di-GMP signaling, is a transcription factor of the LuxR superfamily and was predicted to be a potential receptor of c-di-GMP. To confirm whether the functions of c-di-GMP signaling in H37Rv is same to those in H37Ra, the genes of Rv1357c and Rv0195and the DNA sequences in Rv1354c encoding the GAF or GGDEF domain were deleted from the genome, respectively. The absence of these genes did not affect the infection in human-derived THP-1macrophages and the ability of intracelluar multiplification. However, the interferon IL-8secreted by THP-1cells infected by ARv1354c-GGDEF and ARv1357c were more than that by WT H37Rv during early and middle infection stages.These H37Rv strains were also used to infect C57BL/6mice of SPF grade to estimate their virulence. Infection by WT H37Rv caused mouse spleens to swell rapidly whereas this phenomenon was not observed for ARv1357c. In contrast, the swell of lung due to ARv1357c infection was more serious than that caused by WT H37Rv. According to the pathological analyses of organs, the pathological damages in lungs and spleens caused by ARv1357c after a6-week infection period were more serious than those of WT H37Rv since inflammatory cells were observed more than the latter. On the other hand, ARv1357c in lungs and spleens did not multiply as well as WT H37Rv at early infection stages untill at3w and the colony-forming units （CFUs） of ARv1357c was slightly less than those of WT H37Rv. Intriguingly, ARv1357c isolated from lungs and spleens at10w was nearly unable to recover on7H10plates as no colony was observed after7to8-week growth. In constrast, WT H37Rv isolated formed normal colonies well after3-4w.No obvious difference in the swell of spleens was detected for ARv0195and WT H37Rv. However, ARv0195produced less serious swell in lungs. In contrary to ARv1357c, the pathological damages caused by ARv0195both in lungs and spleens were less serious in comparison with WT H37Rv. Also, ARv0195did not multiply in organs as well as WT H37Rv at early infection stages untill at3w and the CFUs of ARv0195was slightly less than those of WT H37Rv. Similar to ARv1357c, ARv0195isolated from lungs and spleens at10w completely lost the ability to resuscitate on plates.In conclusion, our results obtained umambiguously demonstrated that the c-di-GMP cyclase （DGC） of MTB modulates its c-di-GMP-catalyzing activity by sensing intracellular redox level under hypoxia, and thus regulates by signaling the bio film development, the dormancy under hypoxia and the virulence of MTB. The key factors （DGC, c-di-GMP receptors and its degrading enzyme） in the c-di-GMP signaling pathway is likely essential for MTB dormancy and virulence by modulating a certain level of intracellular c-di-GMP at specific physiological stages. It is anticipated that the three key factors （Rv1354c, Rv0195and Rv1357c） in the c-di-GMP signaling investigated are potential valuable targets for discovery of novel anti-tuberculosis drugs active on dormant MTB.