Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae

Abstract

Sensing and responding to environmental changes is essential for bacteria to adapt and thrive, and nucleotide-derived second messengers are central signaling systems in this process. The most recently identified bacterial cyclic dinucleotide second messenger, 3', 3'-cyclic GMP-AMP (cGAMP), was first discovered in the El Tor biotype of Vibrio cholerae The cGAMP synthase, DncV, is encoded on the VSP-1 pathogenicity island, which is found in all El Tor isolates that are responsible for the current seventh pandemic of cholera but not in the classical biotype. We determined that unregulated production of DncV inhibits growth in El Tor V. cholerae but has no effect on the classical biotype. This cGAMP-dependent phenotype can be suppressed by null mutations in vc0178 immediately 5' of dncV in VSP-1. VC0178 [renamed as cGAMP-activated phospholipase in Vibrio (CapV)] is predicted to be a patatin-like phospholipase, and coexpression of capV and dncV is sufficient to induce growth inhibition in classical V. cholerae and Escherichia coli Furthermore, cGAMP binds to CapV and directly activates its hydrolase activity in vitro. CapV activated by cGAMP in vivo degrades phospholipids in the cell membrane, releasing 16:1 and 18:1 free fatty acids. Together, we demonstrate that cGAMP activates CapV phospholipase activity to target the cell membrane and suggest that acquisition of this second messenger signaling pathway may contribute to the emergence of the El Tor biotype as the etiological agent behind the seventh cholera pandemic.

Keywords: cGAMP; cyclic dinucleotides; pathogenicity island; phospholipid metabolism; second messengers.

Fig. 1.

Overproduction of the cGAMP synthase DncV induces planktonic growth arrest and a small colony phenotype in El Tor V. cholerae. (A) Growth curves of El Tor V. cholerae cultures carrying a P_tac-inducible plasmid encoding dncV (pDncV) or a catalytically inactive mutant (pDncV^mut), grown in the absence (−) or presence (+) of 100 µM IPTG. Each data point represents the mean ± SD of six biological replicates. (B) Colony morphologies of El Tor V. cholerae with pDncV (Left) or pDncV^mut (Middle), or classical V. cholerae strain O395 with pDncV (Right), grown on solid agar plates in the absence (Top) or presence (Bottom) of 100 µM IPTG. (Scale bars: 1 mm.) Two independent plasmid constructs with different copy numbers were tested, and similar results were found (SI Appendix, Fig. S5 and Materials and Methods). Growth curves and colony images are representative of at least three independent experiments.

Fig. 2.

The El Tor VSP-1 gene capV is necessary for cGAMP-induced growth arrest and small colony morphology. (A) Growth curves of El Tor V. cholerae WT and capV::Tn, each carrying pDncV, grown in the absence (−) or presence (+) of 100 µM IPTG. (B) Colony morphologies of the classical V. cholerae strain O395 carrying both pDncV and either an empty vector (Left) or one of two unique El Tor genomic cosmids containing VSP-1 (pCCD7, Middle; pCCD13, Right). Strains were grown on solid agar plates in the absence (Top) or presence (Bottom) of 100 µM IPTG. (Scale bars: 1 mm.) (C) Growth curves of El Tor V. cholerae WT and ΔcapV, each carrying pDncV, grown in the absence (−) or presence (+) of 100 µM IPTG. (D) Colony morphologies of El Tor V. cholerae WT and ΔcapV, each carrying pDncV, grown on solid agar plates in the absence (Top) or presence (Bottom) of 100 µM IPTG. (Scale bars: 1 mm.) (E) Growth curves of El Tor V. cholerae ΔcapV mutants carrying a P_tac-inducible plasmid encoding both dncV and either capV (pCapV-DncV) or a catalytically inactive mutant (pCapV^mut-DncV), grown in the absence (−) or presence (+) of 100 µM IPTG to induce expression of the polycistronic transcript. Each data point in the growth curves represents the mean ± SD of six biological replicates. Growth curves and colony images are representative of at least three independent experiments.

Fig. 3.

cGAMP binding directly induces CapV phospholipase activity. (A–C) Covalent labeling (Top) of the CapV active-site serine by a reactive rhodamine-labeled fluorophosphonate probe (FP-Rh). 1.6 µM His₆-tagged CapV was mixed with both FP-Rh and (A) different concentrations of cGAMP or (B) c-di-GMP or (C) c-di-AMP. (Bottom) Coomassie blue staining of CapV. (D) Thin-layer chromatographic analysis of polar (Left) and neutral (Right) lipids released from the in vitro degradation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by 500 nM purified CapV in the presence of 1 µM cGAMP or other cyclic dinucleotides. DAG, diacylglyceride; FFA, free fatty acid; lyso-PE, lysophosphatidylethanolamine.

Fig. 4.

Overproduction of the cGAMP synthase DncV results in degradation of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) and release of free fatty acids (FFAs) in a CapV-dependent fashion. TLC plates of (A) polar lipids and (B) neutral lipids extracted at 0, 30, and 60 min postinduction with 1 mM IPTG from El Tor V. cholerae WT carrying pDncV or pDncV^mut and from the El Tor V. cholerae ΔcapV mutant carrying pDncV. Plates are representative of three independent experiments. The PE, PG, and FFA standards are dioleoyl-PE, dioleoyl-PG, and linoleic acid, respectively. (C) Quantification (molar percentage of total lipids) by GC of PG, PE, and free fatty acids (FFAs) from A and B. Graphs show the mean of three independent experiments.

Fig. 5.

Overproduction of the cGAMP synthase DncV results in accumulation of 16:1 and 18:1 free fatty acids (FFAs) in the cell. Shown is the proportion (molar percentage) of fatty acid species present in the FFA fractions collected from El Tor V. cholerae WT carrying pDncV (Top) or pDncV^mut (Middle) and from El Tor V. cholerae ΔcapV carrying pDncV (Bottom). Each data point represents the mean ± SD of three independent experiments (*P < 0.01, ***P < 0.0001). ND, not detected; ns, not significant.

Fig. 6.

Overproduction of the cGAMP synthase DncV is detrimental to membrane integrity. Images depict the cell morphology (Top, bright field) and membrane integrity (Bottom, FM 4-64) of El Tor V. cholerae WT carrying pDncV 2 h postinduction with (Right) or without (Left) 100 µM IPTG. Expression of dncV leads to a loss of cellular integrity (Top Right) and altered fluorescent labeling of the cell membrane (Bottom Right). Arrows indicate affected ghost cells. Images are representative of six independent experiments.