Investigation of the Mechanical Properties of the Helicobacter pylori Cell Envelope and Maintenance of Helical Shape by Asymmetric Peptidoglycan Synthesis

Abstract

The human stomach pathogen Helicobacter pylori is a helically-shaped Gram-negative bacterium. While many individual protein contributors to helical shape have been identified and partially characterized, little is known about how these proteins work in concert to achieve helical shape. The structure of the peptidoglycan (PG) cell wall is responsible for H. pylori’s cell shape, and many of the cell shape determining proteins modify or interact with the cell wall, yet how these modifications shape the cell wall is not understood. We demonstrated that cell wall growth is patterned asymmetrically in helical cells, with enhanced synthesis at both the minor and major helical axis areas. We showed that the actin-like cytoskeletal protein MreB has a localization preference for negative Gaussian curvature and that the bactofilin CcmA preferentially localizes to the range of positive Gaussian curvature corresponding to the major helical axis. We demonstrate that in ΔccmA cells, which are gently curved, PG synthesis is still enhanced at negative Gaussian curvature, but there is a dramatic reduction in PG synthesis levels at positive Gaussian curvature. We therefore posit that CcmA-driven enhanced PG synthesis rates at the major helical axis help enable curvature maintenance in the presence of MreB-promoted PG synthesis and that this asymmetric synthesis is one mechanism by which H. pylori maintains helical shape. During these studies we observed that while similar, there are notable differences between PG synthesis labeling patterns with the metabolic probes N-acetylmuramic acid-alkyne (MurNAc-alk) and D-alanine-alkyne (D-Ala-alk). MurNAc-alk is thought to be incorporated through the cytoplasmic steps of the PG biosynthetic pathway, whereas D-Ala-alk is likely incorporated through transpeptidase activity, and thus reports on sites of active crosslinking. Therefore, we performed osmotic and detergent shocks on wild-type and shape mutant H. pylori cells to determine if there is structural heterogeneity in the cell envelope. We observed that, unlike in other rod-like bacteria studied to date, wild-type H. pylori length and width changes in response to turgor pressure modulation via osmotic shock are inversely related, with cells becoming longer and narrower after hyper-osmotic shock and shorter and wider after hypo-osmotic shock. In response to hyper-osmotic shock, the major axis appears to be rigid, with changes in width largely achieved by displacement of the minor helical axis. Detergent treatment with SDS revealed that cells transition through a number of changes before finally shrinking substantially in both length and width, indicating that multiple components of the cell envelope contribute to force homeostasis and that the PG cell wall itself is approximately isotropic with no apparent subcellular heterogeneity.