Functional Analysis of a c-di-AMP-specific Phosphodiesterase MsPDE from Mycobacterium smegmatis

Abstract

Cyclic di‑AMP (c-di-AMP) is a second signaling molecule involved in the regulation of bacterial physiological processes and interaction between pathogen and host. However, the regulatory network mediated by c-di-AMP in Mycobacterium remains obscure. In M. smegmatis, a diadenylate cyclase (DAC) was reported recently, but there is still no investigation on c-di-AMP phosphodiesterase (PDE). Here, we provide a systematic study on signaling mechanism of c-di-AMP PDE in M. smegmatis. Based on our enzymatic analysis, MsPDE (MSMEG_2630), which contained a DHH-DHHA1 domain, displayed a 200-fold higher hydrolytic efficiency (kcat/Km) to c-di-AMP than to c-di-GMP. MsPDE was capable of converting c-di-AMP to pApA and AMP, and hydrolyzing pApA to AMP. Site-directed mutations in DHH and DHHA1 revealed that DHH domain was critical for the phosphodiesterase activity. To explore the regulatory role of c-di-AMP in vivo, we constructed the mspde mutant (Δmspde) and found that deficiency of MsPDE significantly enhanced intracellular C12-C20 fatty acid accumulation. Deficiency of DAC in many bacteria results in cell death. However, we acquired the M. smegmatis strain with DAC gene disrupted (ΔmsdisA) by homologous recombination approach. Deletion of msdisA reduced bacterial C12-C20 fatty acids production but scarcely affected bacterial survival. We also provided evidences that superfluous c-di-AMP in M. smegmatis could lead to abnormal colonial morphology. Collectively, our results indicate that MsPDE is a functional c-di-AMP-specific phosphodiesterase both in vitro and in vivo. Our study also expands the regulatory network mediated by c-di-AMP in M. smegmatis.

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus

“…S. pneumoniae produces a homolog of DhhP named Pde2, which can hydrolyze c-di-AMP and pApA as well as 3′-phosphoadenosine-5′-phosphate (pAp) to AMP and is essential for pathogenesis (24, 29). The Mycobacterium tuberculosis and Mycobacterium smegmatis homologs, MtbPDE and MtPDE, respectively, have been shown to degrade both c-di-AMP and pApA (30–33). A third class of c-di-AMP PDE found in c-di-AMP-producing microorganisms is an HD-domain containing enzyme (17, 34).…”

Too much of a good thing: regulated depletion of c-di-AMP in the bacterial cytoplasm

“…Thus far, these proteins have been characterized in S. pneumoniae, B. burgdorferi, M. tuberculosis , and M. smegmatis [3,7,9,10]. Among those species, only S. pneumoniae also encodes a GdpP homolog, whereas others encode only DhhP.…”

“…Most characterized DhhP proteins degrade both c-di-AMP and 5’-pApA into AMP, as well as c-di-GMP and 5’-pGpG into GMP. Nevertheless, in M. smegmatis , the catalytic efficiency (k cat /k M ) is approximately 200-fold higher for c-di-AMP than for c-di-GMP, indicating that c-di-AMP is the much preferred substrate [10]. Additionally, at least the M. tuberculosis homolog has also been characterized as an NrnA with exonuclease activity on short single stranded nucleic acids, as well as phosphatase activity on pAp [5].…”

“…Thus, in defining a role for DhhP proteins as direct c-di-AMP phosphodiesterases, studies must include thorough kinetic characterization of substrate specificity to further support in vivo changes in c-di-AMP levels mediated by such proteins. Finally, although DhhP is the only DHH-DHHA1 domain protein in M. smegmatis, the dhhP mutant exhibits c-di-AMP levels indistinguishable from WT, indicating the presence of at least another c-di-AMP phosphodiesterase with a different catalytic domain than DHH-DHHA1 [10]. …”

Cyclic dinucleotide (c-di-GMP, c-di-AMP, and cGAMP) signalings have come of age to be inhibited by small molecules

“…111 High c-di-AMP levels have also been shown to trigger stringent response resulting in an increase in the levels of the alarmone (p)ppGpp in S. aureus. 18 Exogenously added c-di-AMP enhanced B. subtilis sporulation 120 while overexpression of MsDisA in M. smegmatis (implying increased c-di-AMP levels) resulted in minute colonies 121 (Fig. 12).…”