Modulation of cAMP levels by a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria

The second messenger, cyclic-AMP (cAMP) is conserved across all taxa of life. It is involved in propagating the signal from environmental stimuli and converting it into a response. In bacteria such as M. tuberculosis (Mtb), P. aeruginosa, V. cholerae and B. pertussis, cAMP has been implicated in virulence, regulation of metabolism and gene expression. Cyclic AMP signalling in mycobacteria is especially complex – with 16 enzymes that produce cAMP in Mtb alone. By discovery of a novel, actinobacteria conserved enzyme that degrades cAMP, we have developed a tool to modulate cAMP levels in mycobacteria. By using a combination of metabolomics, bioenergetics and time-to-kill assays, we show that when this enzyme is overexpressed in the model organism M. smegmatis, there is a 3.3 -fold decrease in intracellular cAMP levels. This was concomitant with altered cell envelope permeability, compromised bioenergetics and most importantly, led to a decrease in the tolerance to various frontline antimicrobials. Taken together, this work provides clear evidence that cAMP is involved in antimicrobial tolerance in mycobacteria and that this may represent a promising new target for antimicrobial development. 6 samples, technical triplicates (i.e. 3 samples) of the empty vector control strain of M. smegmatis and technical triplicates (i.e. an additional 3 samples) of M. smegmatis over-expressing the rv1339 gene.