c-di-GMP (3′-5′-Cyclic Diguanylic Acid) Inhibits Staphylococcus aureus Cell-Cell Interactions and Biofilm Formation

Abstract

Staphylococcus aureus is an important pathogen of humans and animals, and antibiotic resistance is a public health concern. Biofilm formation is essential in virulence and pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. As such, novel antimicrobial approaches are of great interest to the scientific, medical, and agriculture communities. We recently proposed that modulating levels of the cyclic dinucleotide signaling molecule, c-di-GMP (cyclic diguanylate [3,5-cyclic diguanylic acid], cGpGp), has utility in regulating phenotypes of prokaryotes. We report that extracellular c-di-GMP shows activity against human clinical and bovine intramammary mastitis isolates of S. aureus, including methicillinresistant S. aureus (MRSA) isolates. We show that chemically synthesized c-di-GMP is soluble and stable in water and physiological saline and stable following boiling and exposure to acid and alkali. Treatment of S. aureus with extracellular c-di-GMP inhibited cell-to-cell (intercellular) adhesive interactions in liquid medium and reduced (>50%) biofilm formation in human and bovine isolates compared to untreated controls. c-di-GMP inhibited the adherence of S. aureus to human epithelial HeLa cells. The cyclic nucleotide analogs cyclic GMP and cyclic AMP had a lesser inhibitory effect on biofilms, while 5-GMP had no major effect. We propose that cyclic dinucleotides such as c-di-GMP, used either alone or in combination with other antimicrobial agents, represent a novel and attractive approach in the development of intervention strategies for the prevention of biofilms and the control and treatment of infection.

Bacterial c-di-GMP Is an Immunostimulatory Molecule

“…The data presented in the current study show that bacterial c-di-GMP is an immunostimulatory molecule and we have previously demonstrated that c-di-GMP can inhibit bacterial infection (19,21) and has potential in cancer treatment (20). Due to its microbial origin and its effect on the host response, c-di-GMP can be considered as a danger signal or novel pathogen-associated molecular pattern.…”

“…2 shows that pretreatment with c-di-GMP 12 and 6 h before bacterial challenge produces a significant prophylactic effect with a 1.5 and 3.8 log (ϳ10,000-fold) reduction of the mean bacterial CFU in tissues using a 50-and 200-nmol dose, respectively, compared with the untreated control ( p Ͻ 0.05 and p Ͻ 0.001, respectively). Had c-di-GMP only inhibited biofilm formation, as previously shown in our in vitro (19) and in vivo models (21), we would not have expected to find such dramatically reduced numbers of bacterial cells between pretreated and nontreated mice. These results suggest that c-di-GMP might stimulate the innate immune response.…”

“…We have explored the extracellular biological effects of c-di-GMP and its potential use as a therapeutic agent. We have recently shown that extracellular c-di-GMP specifically inhibits S. aureus cell-cell interactions, biofilm formation, and adherence to HeLa epithelial cells in vitro (19). Furthermore, we have shown that c-di-GMP treatment significantly attenuates S. aureus infection in a mouse mastitis model (21).…”

“…We have previously demonstrated that exogenous c-di-GMP significantly reduces in vitro cell-cell interactions and biofilm formation of Staphylococcus aureus, including human methicillin-resistant S. aureus strains and animal clinical isolates (6,19). We have also previously demonstrated in vivo clinical proof-of-concept efficacy that treatment with c-di-GMP (200 nmol) significantly attenuates S. aureus infection and reduces cell numbers (Ͼ10,000-fold; p Ͻ 0.001) in a mouse model of infection (21).…”

Cis-2-decenoic Acid Inhibits S. aureus Growth and Biofilm In Vitro: A Pilot Study

“…In studies of biofilm-reducing compounds, approximate 40% to 80% reductions in biofilm mass were observed [25,27]. With six concentrations of C2DA studied, given the approximate 20% standard deviation (SD), we could detect a difference of 75% of control values with 80% power using ANOVA analysis with three wells for the biofilm inhibition studies.…”