Functional Analysis Of The C-di-AMP Metabolism Genes Harbored In Streptococcus Suis Serotype 2

Cyclic diadenosine monophosphate（c-di-AMP）, is a new second messenger found recently in bacteria. The level of c-di-AMP in bacteria is regulated by the activities of diadenylate cyclase（DAC） and phosphodiesterases（PDE）. c-di-AMP appears to regulate various aspects of bacterial physiology, including cell growth, cell wall homeostasis and virulence. Streptococcus suis is a major worldwide swine pathogen. Streptococcus. suis serotype 2（SS2）, among the identified 33 serotypes（types l 31, 33 and l/2） according to capsular polysaccharides, is considered the most virulent and is frequently recovered from diseased animals, causing arthritis, endocarditis, meningitis, pneumonia, and septicemia. It has been reported that many virulence related factor have been identified in SS2, however, the existent and biofunction of the c-diAMP, as an important second messenger, and its metabolic pathway in SS2 has not yet been explored. In this study, using SS2 high virulent strain HA9801 as the research object, we identified the existent of the c-di-AMP metabolic pathway, for the first time, and explored the role of it played in physiology and virulence of SS2.To determine whether c-di-AMP was produced in SS2 HA9801, the cell extract was injected and separated by HPLC. A peak at the same retention time with the c-diAMP standard was detected. The relevant fractions were collected and verify the presence of c-di-AMP by ESI-MS. These suggested that SS2 HA9801 synthesized cdi-AMP in vivo. The identified of c-di-AMP will provide a solid basis for future research concerning the role of c-di-AMP in SS2. Further studies showed that the level of c-di-AMP in SS2 slightly raised along with its growth, and reach to a stable level after the exponential phase according to the calibration curve of c-di-AMP standard.Base on the bioinformatics analysis, we identified the c-di-AMP metabolism genes（dacA and gdpP） in SS2 according to the 98HAH33 genomic sequence（NC₀09443.1） published in NCBI. We cloned these two genes using the genome DNA of SS2 HA9801 strain as template, and the bioinformatics analysis showed that the dacA gene encode a 283-amino-acid protein product possesses 3 transmembrane domains and a DisA_N domain which is critical for the diadenylate cyclase activity. The gdpP gene encode a 654-amino-acid protein product possesses 2 transmembrane domains, a PAS domain, and a DHH/DHHA1 domain which is critical for the phosphodiesterase activity which are conserved in the GdpP homologues. Both of these two proteins exhibit highly similarity to those proteins from other gram-positive bacteria. In addition, the results of RT-PCR showed that both dacA and gdpP are expressed together with their downstream genes as one transcript.To verify the function of the predicted genes, we expressed and purified the DacA and GdpP encoded by the two predicted genes. The enzymatic activity assay using HPLC and ESI-MS showed that DacA was a diadenylate cyclase, for the recombinant DacA 99-283 converted ATP into c-di-AMP in vitro. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+ or Co2+ and preferred Mg2+ as co-factors. The recombinant GdpP73-654 protein hydrolyzed the c-di-AMP to pApA and preferred Mn2+ as co-factors. Both of these two proteins exhibited more active at a basic pH rather than at an acidic pH. We also found that DGA and RHR motifs are essential for the diadenylate cyclase activity, especially the RHR motif is the ATP binding site of DacA, and the DHH/DHHA1 domain is the critical region for the phosphodiesterase activity.To further determine the biofunctional of the c-di-AMP and its metabolism genes in SS2, we tried to construct the dacA and gdpP mutant strains. To delete the genes without affecting the transcription of their downstream genes, the in-frame deletion allele of each gene was carried out. Finally, the gdpP inframe deletion mutant was constructed successfully confirmed by clony PCR and direct DNA sequencing, while we can’t get the dacA deletion mutant, for this gene seems to be essential for viability of SS2. For functional complementation, the DNA fragment including the gdpP coding region plus its upstream promoter sequence was amplified from the SS2 HA9801 genome DNA. Then the product was cloned into E. coli-S. suis shuttle vector pSET2 s, and electrotransformed into the gdpP mutant to obtain the complemented strain.To test the effects of gdpP deletion on SS2, the biological properties of the wild type strain and the mutant strain were compared under the same conditions. Gram staining analysis showed that the gdpP mutant tends to aggregate into clusters, exhibit abnormal morphology compared with the wild type strain. Scanning electron microscope and transmission electron microscopy showed that the deletion of gdpP led to cell expansion, and some of the mutant lost the typical shape characteristic of SS2. Growth curves showed that gdpP mutant exhibited the lagged logarithmic phase compared with the wild type strain. Biofilm formation by the gdpP mutant was significantly increased by 1.86-fold, while disruption of gdpP significantly reduced the hemolytic activity in culture supernatant fluids. In addition, deletion of the gdpP gene significantly reduced adherence to and invasion of HEp-2 cells, to 36.8% and 57.0%, respectively, compared with the wild strain. In murine infection models, the LD50 of the mutant strain（7.23 × 108） was increased compared with the wild-type strain（3.00 × 107）, and the survival rates of mice infected with the mutant strain were increased compared with the wild-type strain. The numbers of wild type strain recovered from specific organs was higher（⁵.0 log CFU/g） compared with gdpP mutant（³.9 log CFU/g）. Besides, pathological examination showed that specific organs including brain, lung and spleen suffered less damage in the mutant-infected group than in which the wild type strain infected group. All these findings revealed a significant contribution of gdpP and c-di-AMP level to the biology and virulence of SS2.To explore how the gdpP mutation caused so many phenotype alterations in SS2, we determined the global gene transcription profiles changes between gdp P mutant and wild type strain by RNA-seq and qPCR. Our results showed that 214 genes, represented on the RAN-seq assay were differentially transcribed in the gdpP mutant strain compared to the WT strain. All these altered transcripts encoding proteins associated with information storage and processing, protein biosynthesis and modification, virulence and so on. Suggesting the c-di-AMP is an important second messenger and plays a critical role in global regulatory networks in SS2.To further understand the accurate mechanism by which c-di-AMP plays in SS2, it is crucial to identify the c-di-AMP binding proteins in SS2 HA9801. We established the method to separate the c-di-AMP binding proteins base on the 2’-AHC-c-diAMPagarose, using the purified the Bacillus subtilis DisA protein which possesses the c-diAMP binding ability and the BSA as the positive and negative control respectively. Bacterial lysates of SS2 HA9801 were then applied to purify any c-di-AMP binding proteins. 11 apparent protein bands were detected in SDS-PAGE, and the designated bands were then in-gel tryptic digested and analyzed with LC-MS/MS. All MS/MS spectra obtained from the experiment were used to search the NCBI nonredundant database using the Mascot database, and each protein was functional annotated. The information we got are expected to significantly contribute to our further understanding of the regulatory roles of c-di-AMP in SS2.In conclusion, we identified the existence of c-di-AMP and its metabolism genes in SS2. The alterations of phenotypes, virulence and gene transcription profiles in the gdpP mutant strain indicated that c-di-AMP plays an important role in global regulatory networks in SS2 pathogenesis.