Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

Abstract

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.

Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain

“…1g). The excellent fit of c-di-GMP within the motif also explains why the MshEN domain cannot bind cGMP, GMP, GDP or GTP, or the adenine base-containing nucleotides such as cAMP, ATP, ADP and AMP, as has been reported previously5 and also confirmed here using differential scanning fluorimetry (DSF, Supplementary Fig. 2).…”

“…The plasmid pVL393- mshE 5, containing the wild-type mshE structural gene, was used as a template to introduce point mutations at positions 10 (L10A), 11 (G11I), 58 (L58A) and 54–58 (L54A–L58A). The resulting plasmids pVL393- mshE -L58A and pVL393- mshE -L54A-L58A were used as templates to introduce an additional point mutation at position 10, resulting in constructs pVL393- mshE -L10A-L58A and VL393- mshE -L10A-L54A-L58A, respectively.…”

Versatile modes of cellular regulation via cyclic dinucleotides

“…biotinylated) dinucleotide homologues as capture compounds for the selective pull-down of CDN-binding proteins 17,18,43,44 . In addition, the D ifferential Ra dial C apillary A ction of L igand A ssay (DRaCALA) – a method relying on labeled, protein-bound ligand retention upon spotting onto a nitrocellulose membrane – has been used with both purified proteins and expression library lysates for the systematic identification of novel CDN sensors 18,45,46 .…”

“…Examples include Type IV pili/Type II SS-associated MshE homologues from V. cholerae and P. aeruginosa, the flagellar export ATPase FliL from diverse bacteria and rotary ATPases HrcN and ClpB2 associated with pseudomonad Type III and Type VI SS, respectively 46,66 (Figure 2). Residues key for c-di-GMP recognition have been identified for some of these targets and the recognition mechanisms appear to differ significantly from that of the ATPase FleQ 59 .…”

Cyclic Dimeric Guanosine Monophosphate: Activation and Inhibition of Innate Immune Response

“…Recently, by using liquid chromatography and mass spectrometry, we determined the concentrations of c-di-GMP in bacterial culture supernatants to be 1.042 ± 0.12 mM for E. coli cells and 0.568 ± 0.06 mM for M. tuberculosis [24]. In addition, c-di-GMP was found to regulate type III and type VI secretion systems in Pseudomonas aeruginosa [39, 40]. Therefore, growing evidence suggests that bacteria can secrete c-di-GMP.…”