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.

Construction and Analysis of the Complete Genome Sequence of Leprosy Agent Mycobacterium lepromatosis

“…The presence of three copies of its gene hints more c-di-GMP metabolism in M. lepromatosis for survival and/or pathogenicity in view that deletion of this gene in M. tuberculosis affects its dormancy and pathogenicity (39,40). In M. leprae, studies have also suggested a potential role of diguanylate cyclase in the signaling response for intracellular survival (41). In other pathogens, a strategy has been designed to intercept c-di-GMP signaling pathways by directly targeting this second messenger as a way of controlling pathogen survival, growth or biofilm formation (42).…”

Disruption of c-di-GMP Signaling Networks Unlocks Cryptic Expression of Secondary Metabolites during Biofilm Growth in Burkholderia pseudomallei

“…We also evaluated the activity of these genes using a heterologous approach to evaluate the effect on c-di-GMP signaling-mediated phenotypes in Pseudomonas aeruginosa PAO1 ( 22 – 24 ). Inducible expression of cdpA or cdpA -I2285 in either P. aeruginosa PAO1 (see Fig.…”

Motility and biofilm production involved in the interaction of phosphate solubilizing endophytic strains with peanut, maize and soybean plants

“…Considering twitching motility, several studies determine that this bacterial capacity is possible to be shown through staining with Crystal Violet of bacterial growth that occurs at the interface between the agar medium and the Petri dish (Rotcheewaphan et al, 2016;Tomada et al, 2016). In addition, studies with mutants of Azoarcus spp., defective in the production of type IV pili, demonstrated the importance of this cell structure in twitching motility, not only to join the surface of the plant but also to colonize the inside of the root (B€ ohm et al, 2007).…”