Transition state analogs of 5'-methylthioadenosine nucleosidase disrupt quorum sensing

Abstract

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme involved in S-adenosylmethionine-related quorum sensing pathways that induce bacterial pathogenesis factors. Transition state analogs MT-DADMe-Immucillin-A, EtT-DADMe-Immucillin-A and BuT-DADMe-Immucillin-A are slow-onset, tight-binding inhibitors of Vibrio cholerae MTAN (VcMTAN), with equilibrium dissociation constants of 73, 70 and 208 pM, respectively. Structural analysis of VcMTAN with BuT-DADMe-Immucillin-A revealed interactions contributing to the high affinity. We found that in V. cholerae cells, these compounds are potent MTAN inhibitors with IC(50) values of 27, 31 and 6 nM for MT-, EtT- and BuT-DADMe-Immucillin-A, respectively; the compounds disrupt autoinducer production in a dose-dependent manner without affecting growth. MT- and BuT-DADMe-Immucillin-A also inhibited autoinducer-2 production in enterohemorrhagic Escherichia coli O157:H7 with IC(50) values of 600 and 125 nM, respectively. BuT-DADMe-Immucillin-A inhibition of autoinducer-2 production in both strains persisted for several generations and caused reduction in biofilm formation. These results support MTAN's role in quorum sensing and its potential as a target for bacterial anti-infective drug design.

Figure 1

Role of MTAN in bacterial utilization of S-adenosylmethionine (SAM). The scheme shows the pathways connecting DNA methylation (red box), polyamine synthesis (blue box), autoinducer production (yellow box), and methionine and adenine salvage. AHL synthase catalyzes the transfer of the amino acid moiety of SAM to an acyl acceptor to produce homoserine lactones in the synthesis of AI-1 molecules, and MTA as byproduct. In methyltransferase reactions, SAM produces SAH which is a precursor in the tetrahydrofuran synthesis of AI-2 molecules (shown here as furanosyl boron diester, 14). Blocking MTAN activity is expected to cause accumulation of MTA, resulting in product inhibition of AI-1 production by AHL synthase. In addition, inhibition of MTAN can directly block the formation of S-ribosylhomocysteine (SRH, 15), the precursor of AI-2. AI-1 and AI-2 are autoinducers used in bacterial quorum sensing, and MTAN offers a means to block formation of these signaling molecules.

Figure 2

The reaction catalyzed by MTAN with MTA as substrate. (a, top) shows a dissociative transition state for E. coli with ribooxacarbenium ion character. Structures of stable analogues for an early dissociative transition state (ImmucillinA), and a late dissociative transition state (DADMe-ImmucillinA) depict differences in bond distances between the adenine leaving group and the ribosyl group, as well as charge localization (a, bottom). (b) Shown is the structure of S-substituted DADMe-ImmucillinA, along with MT-, EtT- and BuT- substituents.

Figure 3

Crystal structure of VcMTAN in complex with BuT-DADMe-ImmA. (a) Overall structure of the VcMTAN structure showing the asymmetric unit content with the inhibitor BuT-DADMe-ImmA bound in the active sites. (b) The active site of the VcMTAN with a 2Fo − Fc map contoured at 1.2σ surrounding the BuT-DADMe-ImmA inhibitor and the proposed nucleophilic water molecule. (c) Space filling picture of the active site of VcMTAN with BuT-DADMe-ImmA in the active site. Grey represents hydrophobic regions of the protein which interact with hydrophobic parts of the inhibitor. The red color shows parts of the protein that contain charged residues interacting with polar groups of the inhibitor, while green represents loop regions. (d) Schematic drawing of the BuT-DADMe-ImmA inhibitor bound in the active site of VcMTAN showing catalytic contacts.

Figure 4

Effect of BuT-DADMe-ImmA on autoinducer-2 production in pathogenic E. coli and V. cholerae upon short-term and long-term inhibitor treatment, and on static biofilm formation. (a) E. coli O157:H7 ± 0.5 μM BuT-DADMe-ImmA, and an MTAN⁻strain were grown static in AB medium for >5 generations before assaying the spent medium for autoinducer-2 production. (b) E. coli O157:H7, and (c) V. cholerae N16961 grown shaken in LB for 26 generations, and autoinducer-2 in the spent media was measured. Cultures were prepared in triplicate, and data represent mean values ± s.d. from at least 6 replicates. Biofilm formation studies on (d) E. coli O157:H7, and (e) V. cholerae N16961, ± 1 μM BuT-DADMe-ImmA grown static in LB medium at 25 °C for 24 hours on 96-well format. The observed difference in biofilm formation due to BuT-DADMe-ImmA was statistically significant at t = 5.044, ***p < 0.001, d.f. = 14 for E. coli; and t = 26.689, p < 0.001, d.f. = 14 for V. cholerae.