The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability
- 740 Downloads
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.
Keywordsc-di-GMP Diguanylate cyclase Biofilm formation Antimetabolite drugs Crohn’s disease Azathioprine
We thank Grant Burgess and Flavio Caprioli for a critical reading of the manuscript, Urs Jenal and Holger Sondermann for providing the plasmids for PleD, WspR, and RocR overexpression, and Silvia Fernicola for help with the in vitro inhibition studies. Funding for this study was provided by the Italian Foundation for Research on Cystic Fibrosis (project FFC#13/2009, with the contribution of Delegazione Novara and Delegazione Cosenza-2), by the CHEM-PROFARMA-NET Research Program (Project RBPR05NWWC_004), and PRIN/FIRB Research Programs (Projects 20094BJ9R7 and RBFR10LHD1) of the Italian Ministry for University and Research.
- Bardonnet N, Blanco C (1992) uidA antibiotic resistance cassettes for insertion mutagenesis, gene fusion and genetic constructions. FEMS Microbiol Lett 93:243–248Google Scholar
- Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B, Yantiss R, Orsi RH, Wiedmann M, McDonough P, Kim SG, Berg D, Schukken Y, Scherl E, Simpson KW (2007) Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. ISME J 1:403–418PubMedCrossRefGoogle Scholar
- Clinical and Laboratory Standards Institute (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 7th edn. Approved standard M7-A7. Clinical and Laboratory Standards Institute, WayneGoogle Scholar
- Dignass A, Van Assche G, Lindsay JO, Lémann M, Söderholm J, Colombel JF, Danese S, D'Hoore A, Gassull M, Gomollón F, Hommes DW, Michetti P, O'Morain C, Oresland T, Windsor A, Stange EF, Travis SP, European Crohn's and Colitis Organisation (ECCO) (2010) The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management. J Crohns Colitis 4:28–62PubMedCrossRefGoogle Scholar
- Kulasakara H, Lee V, Brencic A, Liberati N, Urbach J, Miyata S, Lee DG, Neely AN, Hyodo M, Hayakawa Y, Ausubel FM, Lory S (2006) Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence. Proc Natl Acad Sci USA 103:2839–2844PubMedCrossRefGoogle Scholar
- Levesque BG, Kane SV (2011) Searching for the delta: 5-aminosalicylic acid therapy for Crohn's disease. Gastroenterol Hepatol (NY) 7:295–301Google Scholar
- Robbe-Saule V, Jaumouille V, Prevost MC, Guadagnini S, Talhouarne C, Mathout H, Kolb A, Norel F (2006) Crl activates transcription initiation of RpoS-regulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium. J Bacteriol 188:3983–3994PubMedCrossRefGoogle Scholar
- Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964PubMedCrossRefGoogle Scholar
- Tiede I, Fritz G, Strand S, Poppe D, Dvorsky R, Strand D, Lehr HA, Wirtz S, Becker C, Atreya R, Mudter J, Hildner K, Bartsch B, Holtmann M, Blumberg R, Walczak H, Iven H, Galle PR, Ahmadian MR, Neurath MF (2003) CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest 111:1133–1145PubMedGoogle Scholar
- Wassmann P, Chan C, Paul R, Beck A, Heerklotz H, Jenal U, Schirmer T (2007) Structure of BeF3−–modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition. Structure 15:915–927Google Scholar
- Whitney JC, Howell PL (2012) Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria. Trends Microbiol. 2012. doi: 10.1016/j.tim.2012.10.001