A Bacterial Cell Shape-Determining Inhibitor

Abstract

Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4 and its C. jejuni homologue Pgp1 cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap, with a Ki value of 1.5 μM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials against these human pathogens.

Figure 1

A) Peptidoglycan trimming and the reaction catalyzed by Csd4. The inset shows the structure of meso-Dap. B) The proposed catalytic mechanism for the Csd4 reaction.

Figure 2

Comparison of structures of Csd4 in complex with a N-acetyltripeptide substrate and inhibitor 1. A) The structure of N-acetyltripeptide complexed with Csd4 (produced using PDB ID: 4WCN). The substrate carbon atoms are in dark grey; selected Csd4-substrate interactions are shown as yellow dotted lines; zinc ligands are colored purple; carbon atoms of residues interacting with the tripeptide are colored white; the predicted catalytic water is red; zinc is grey. B) Structure of the Csd4-inhibitor 1 complex. The bound inhibitor (carbon atoms in blue) with key active site residues are highlighted. Carbon atoms of selected amino acid residues that interact with the inhibitor are shown in teal. C) Superposition of the structures of Csd4 with bound substrate and inhibitor 1. The substrate, inhibitor 1 and zinc ligands are shown as in panels A and B. D) Top, the initial omit F_o−F_c map contoured at 3.5 σ prior to modelling of the inhibitor, and below, the final refined 2F_o−F_c map of the inhibitor contoured at 1.5 σ. In both maps, the final refined inhibitor model is included for visualization purposes. The (S)-configuration of the inhibitor stereocenter that is closest to the phosphinate is labeled. In all panels, oxygen, nitrogen and phosphorus atoms are shown in red, blue and orange, respectively.

Figure 3

A metal-coordinated tetrahedral intermediate and the corresponding phosphorous-based inhibitors.

Figure 4

A Dixon plot showing the inhibition of Csd4 by inhibitor 1. The value of [I'] was adjusted to account for enzyme-bound inhibitor.

Figure 5

Inhibitor 1 alters H. pylori cell shape. A) Phase contrast images of H. pylori (KBH19) treated for 10 hours without (blue, left) or with 2.1 mM inhibitor 1 (red, right). B) Scatterplot of 100–200 cell contours per condition from phase contrast images of cells grown for 10 hours with 2.1 mM inhibitor 1 (red) or without (blue). Axis length is plotted on the x-axis and side curvature is plotted on the y-axis. C) Smooth histogram of population side curvature values for cells treated for 10 hours without (blue) and with 2.1 mM inhibitor 1 (red). Treated cells have significantly lower side curvatures than u––––ntreated cells (p<0.00001).

Figure 6

C. jejuni wild type 81–176 and ΔkpsM show cell straightening in the presence of inhibitor 1. DIC microscopy images of C. jejuni 81–176 (A) and the acapsular ΔkpsM (D) without inhibitor (left) and treated with 2.3 mM inhibitor for 24h (right). Scatter plots arraying cell length (x-axis, µm) and side curvature (y-axis, arbitrary units) for 81–176 (B) and ΔkpsM (E) grown for 24 h with and without inhibitor 1. Each contour represents the morphology of a single cell from a 1000× DIC image as determined using CellTool software. Smooth histograms displaying population side curvature (x-axis, arbitrary units) as a density function (y-axis) for 81–176 (C) and ΔkpsM (F) grown for 24 h with and without inhibitor 1. Kolmogorov–Smirnov statistical comparisons of population cell side curvature distributions indicate that treated cells have significantly lower side curvatures than untreated cells (81–176, p<0.00001; ΔkpsM, p<0.00001, with p<0.05 indicating significance).

Scheme 1

The synthesis of inhibitor 1.