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Probing the Role of Peptidoglycan Metabolism in Helicobacter pylori's Helical Shape

Helical cell shape is important for Helicobacter pylori's ability to colonize the human stomach. Cell shape in this Gram‐negative pathogen is determined by the cell wall, which is composed of peptidoglycan (PG), a polymer of disaccharides with an attached pentapeptide stem that can join glycan strands by crosslinking with an adjacent peptide stem. Maintaining helical shape requires the activity of two endopeptidases, two carboxypeptidases, and several non‐enzymatic proteins. This suggests that structural modification of the PG is essential for shape maintenance, but it is unclear whether structural modification alone is sufficient or if modifications act in concert with PG synthesis and/or turnover. The most widely‐adopted metabolic probe for labeling new PG synthesis is either a small fluorophore or clickable alkyne conjugated to D‐alanine, which is present in the two terminal positions in the PG peptide stem. However, this probe appears to be mainly incorporated through transpeptidation reactions, which do occur at new PG synthesis sites, but may also occur during synthesis‐independent modification of the cell wall. We generated a strain of H. pylori that expresses Pseudomonas putida N‐acetylmuramic acid (MurNAc ) recycling enzymes AmgK and MurU and is capable of incorporating the newly‐developed MurNAc‐alkyne probe into the cell wall. We are employing quantitative analysis of 3D structured illumination microscopy (SIM) images to define the different regions of the helical cell using Gaussian curvature and to measure whether D‐alanine‐alkyne and MurNAc‐alkyne are preferentially enriched at positive or negative curvature (major or minor helical axis of a helical cell, respectively). Preliminary results indicate that the two probes show different curvature enrichment and suggest that crosslinking activity occurs independently of new PG synthesis and/or crosslinking rates during synthesis differ at different parts of the cell. These results suggest a role for spatial regulation of both structural variation and new PG synthesis in maintaining H. pylori's helical cell shape. Furthermore, these results demonstrate the value of employing both metabolic probes to investigate cell growth and shape maintenance.