The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability

In Gram-negative bacteria, production of the signal molecule c-di-GMP by
diguanylate cyclases (DGCs) is a key trigger for biofilm formation,
which, in turn, is often required for the development of chronic
bacterial infections. Thus, DGCs represent interesting targets for new
chemotherapeutic drugs with anti-biofilm activity. We searched for
inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in
biofilm formation and production of virulence factors, using a set of
microbiological assays developed in an Escherichia coli strain
expressing the wspR gene. We found that azathioprine, an
immunosuppressive drug used in the treatment of Crohn's disease, was
able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells.
However, in vitro enzymatic assays ruled out direct inhibition of WspR
DGC activity either by azathioprine or by its metabolic derivative
2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of
5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an
enzyme involved in purine biosynthesis, which suggests that inhibition
of c-di-GMP biosynthesis by azathioprine may be due to perturbation of
intracellular nucleotide pools. Consistent with this hypothesis, WspR
activity is abolished in an E. coli purH mutant strain, unable to
produce AICAR transformylase. Despite its effect on WspR, azathioprine
failed to prevent biofilm formation by P. aeruginosa; however, it
affected production of extracellular structures in E. coli clinical
isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in
this bacterium. Our results indicate that azathioprine can prevent
biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis
and suggest that such inhibition might contribute to its
anti-inflammatory activity in Crohn's disease.