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, inhibiting cell shape determinant proteases and altering cell shape, thereby affecting the pathogenic attributes of these organisms. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials against these human pathogens.

Genomic variations leading to alterations in cell morphology of Campylobacter spp

“…These targets are broad-spectrum in the sense that endo- and carboxypeptidases are common throughout the bacterial kingdom5657, but many PG peptidases are redundant5859 and therefore detrimental effects might be compensated in other species and it may also be possible to design narrow-spectrum options that specifically target Pgp1 or Pgp2. Intriguingly, Liu et al 60. recently reported a bacterial cell shape-determining inhibitor of H. pylori Csd4 (homologue of Pgp1 in C. jejuni ) that causes significant cell straightening of H. pylori and a diminished, yet observable, effect on the morphology of C. jejuni .…”

Structural Basis of the Heterodimer Formation between Cell Shape-Determining Proteins Csd1 and Csd2 from Helicobacter pylori

“…A recent study described the synthesis of a phosphinic acid-based inhibitor against Csd4 from H . pylori [ 60 ]. It demonstrated that the hydrophilic small-molecule inhibitor of Csd4 can cross the outer membrane of H .…”

Morphology of Helicobacter pylori as a result of peptidoglycan and cytoskeleton rearrangements

“…One of the ways to reduce the pathogenicity of H. pylori is the possibility of disrupting helical cell formation. An example of a substance with such properties is a phosphonic acid-based pseudopeptide, which has the ability to bind to the active site of Csd4 (or Pgp1 in Campylobacter jejuni ) and mediate protein activity disturbances [ 89 ]. The millimolar concentrations of this compound contributed to the straightening of H. pylori and C. jejuni cells.…”