Functional analysis of cyclic dinucleotide-binding proteins in prokaryotic and eukaryotic cells

Abstract

Cyclic dinucleotides (CDNs) are a set of intracellular signaling molecules with various physiological roles in both prokaryotes and eukaryotes. CDN acts as a second messenger, transmitting receptor-activated signals to downstream signaling pathways. Bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) is a well-characterized second messenger in a bacterial system, which is synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). c-di-GMP regulates multiple intracellular biological functions, such as biofilm formation, virulence and motility, via binding its downstream effectors. Several c-di-GMP binding proteins have been identified, mainly associated with the PilZ domain, DGCs, and PDEs. However, whether c-di-GMP is involved in other cellular signaling pathways remains elucidated. Stenotrophomonas maltophilia is an opportunistic bacterium considered an “emerging pathogen of concern” and the third most common non-fermentative bacterium isolated in clinical settings. This pathogen is harmful to patients with conditions such as acquired immunodeficiency syndrome (AIDS), cystic fibrosis (CF), and those receiving immunosuppressive treatments or long-term intensive care unit (ICU) stays. Therefore, we used S. maltophilia as the study subject to identify the novel binding proteins to c-di-GMP. Our study found that c-di-GMP can bind to Superoxide dismutase A (SodA) and ATP-dependent Clp protease proteolytic subunit (ClpP) in vitro. SodA primarily neutralizes superoxide to execute detoxification functions within the cells. We defined that SodA is a novel c-di-GMP effector that regulates oxidative stress tolerance in a c-di-GMP-dependent manner. SodA enzymatic activity was reduced and the secondary structure was altered when bound with c-di-GMP. Our research offers new understanding of how bacteria initiate a stress response in response to elevated levels of bacterial second messengers. ClpP functions as a protease that degrades the misfolded or damaged proteins, which is essential in protein quality control and intracellular process regulation. Our study found that ClpP peptidase activity was enhanced by directly binding with c-di-GMP in vitro. ClpP is also involved in biofilm formation by regulating the polysaccharide metabolism. This provides a new insight into how c-di-GMP regulates biofilm formation via intracellular protease. In mammalian cells, a hybrid CDN, 2’3’-cyclic GMP-AMP (cGAMP), has been characterized as a second messenger involved in the cGAS-STING pathway to trigger downstream type I interferon production. γ-IFN-Inducible protein 16 (IFI16) is an innate immune sensor that stimulates innate immune response, which mediates the promotion of interferon production and inflammasome activation. We have characterized IFI16 as a new cGAMP binding protein, promoting the production of type I interferon and interferon-stimulated genes (ISGs) as well as other pro-inflammatory cytokines and chemokines. Our study has revealed new mechanistic details of cGAMP dependency of IFI16-associated innate immune response.