Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds

Abstract

With antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two-component system. Two-component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics and also to trigger production of virulence factors. Drugs that target the response regulator portion of two-component systems represent a potent new approach so far unexploited. Here, we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initially present the structure of the N-terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with2-aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.

Fig. 1

Structure of Francisella response regulator, QseBN. The N-terminal domain of QseB is composed of two monomers, blue and green. Secondary structure elements are labeled.

Fig. 2

QseB binds 2-AI compounds. A) Chemical structure of AGL-600. B) Chemical structure of AGL-726. C) Simulated ITC data showing AGL-600 binds QseB with a K_d of 200.6 ± 9.8 µM. D) Thermal shift data showing dose-dependent change in T_m of QseB with addition of AGL-726.

Fig. 3

Molecular docking of AGL-726 to a model of QseB. A) Full-length model of QseB based off of homologous crystal structures. One monomer (blue) takes on the “tucked” conformation with N-terminal domain in dark blue and C-terminal domain in light blue. The second monomer (green) is in the “extended” conformation with N-terminal domain in dark green and C-terminal domain in light green. B) Surface representation of AGL-726 (orange) docked to the “tucked” (blue) and “extended” (green) conformations. C) Close-up view of binding pocket of the “tucked” conformation. Yellow dashes representing H-bonding distances between AGL-726 and side chains. D) Close-up view of “extended” conformation binding site with yellow dashes representing potential H-bonding.

Fig. 4

Intramacrophage survival assay. F. tularensis novicida that was exposed to AGL-600 and AGL-726 prior to infection show a decrease in survivability within J774A.1 macrophages after a 12 h incubation. Assays were conducted at a multiplicity of infection of about 50:1, bacteria to macrophages.

Fig. 5

Proposed mechanisms for 2-AI binding. Response regulator shown as a blue cartoon and 2-AI as an orange square. 2-AIs either coerce the response regulator to favor a “tucked” conformation (left-hand path), hindering DNA-binding activity, or they bind in a manner that pulls the domains apart into the “extended” conformation (right-hand path), reducing protein stability.