Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

Abstract

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

Fig. 1.

Bioinformatics analyses of putative DGC and PDE of M. leprae. (a) ML1750c (623 aa) (gi|15827936|NP_302199) is a hybrid protein containing both GGDEF and EAL motifs and an N-terminal GAF sensory domain. (b) ML1419c (563 aa) (gi|15827746|NP_302009) contains a GGDEF motif and three consecutive PAS sensory domains upstream to GGDEF domain. (c) ML0397c (602 aa) (gi|15827122|NP_301385) possesses a GGDEF motif, an N-terminal PAS sensor domain and 10 transmembrane α-helices (red rectangles). (d) ML1752c (302 aa) (gi|15827938|NP_302201) has a single EAL motif and lacks a sensory domain. Homologues of ML1750c are produced in both M. tuberculosis and M. smegmatis and a homologue of ML1752c is identified in M. tuberculosis. Numbers indicate amino acid positions as reported by CDD NCBI. (e) Alignment of conserved DGC domains of M. leprae proteins ML0397c, ML1419c and ML1750c. The conserved I-site, RxxD motif of ML0397c and ML1419c is highlighted in grey. The conserved A-site, RxGGDEF motif, is present in all proteins. Conserved amino acids involved in enzymatic activity are highlighted in yellow.

Fig. 2.

Expression of ml1419c from M. leprae in P. aeruginosa PAO1. Recombinant P. aeruginosa PAO1 containing ml1419c, ml1419c_ΔGGDEF or the pJN105 vector (VC) were grown in the presence (+) or in the absence (−) of 0.2 % l- arabinose. Whole cell lysates (5 µg) of the recombinant strains were analysed by SDS-PAGE with Coomassie blue staining (a) and Western blot (b). Purified recombinant ML1419c produced in E. coli was used as a positive control (lane P) for Coomassie blue staining (500 ng) and Western blot (25 ng).

Fig. 3.

Colony morphology of P. aeruginosa PAO1 and recombinant strains. Strains were grown on VBMM agar containing Congo red, brilliant blue and 1 % l-arabinose. Rugose colonies were observed in P. aeruginosa expressing tpbB and ml1419c. PAO1 wild-type, P. aeruginosa expressing ml1419c_ΔGGDEF and the vector control (VC) form round colonies with smooth surfaces. Scale bar corresponds to 1 mm.

Fig. 4.

P. aeruginosa expressing ml1419c suppresses motility and enhances biofilm formation. (a) Swimming and (b) twitching motility were suppressed in P. aeruginosa expressing tpbB and ml1419c as compared to the wild-type PAO1 strain and vector control (VC) in the presence of 0.2 % l-arabinose. Twitching and swimming motility were restored in P. aeruginosa expressing mutated ml1419c (ml1419c_ΔGGDEF). The diameters (mm) of (a) swim zones and (b) twitching zones were measured for four replicates of each strain and the mean was determined, *P<0.0001. (c) Biofilm formation as measured by crystal violet binding was increased in P. aeruginosa expressing tpbB and ml1419c. Biofilm formation was abrogated in P. aeruginosa expressing ml1419c_ΔGGDEF. The mean was determined from six replicates. *P<0.0001.

Fig. 5.

Cyclic di-GMP detection and relative quantification of cyclic di-GMP in recombinant P. aeruginosa strains. The LC–MS extracted ion chromatogram of cyclic di-GMP (m/z 691.102) in extracts of recombinant P. aeruginosa strains; (a) vector control (VC), (b) ml1419c and (c) ml1419c_ΔGGDEF. [¹³C]Adenosine was applied as an internal standard (retention time 7.702 min, m/z 269.1065). (d) The relative quantification demonstrated a significant increase in the abundance of cyclic di-GMP produced in P. aeruginosa expressing ml1419c as compared to VC and P. aeruginosa expressing ml1419c_ΔGGDEF, *P<0.0001. Experiments were performed with three biological and three technical replicates.