Mapping nanostructural changes in E.coli Peptidoglycan

E.coli is a rod-shaped Gram-negative bacterium whose shape is maintained by a biopolymer known as Peptidoglycan (PG)¹. The chemical composition of PG is well understood² but the following questions remain unanswered; 1) what is the detailed molecular organization of the PG; 2) is its organization location-dependent or not; 3) In the case of antibiotic-induced shape change and death, what happens to the PG organization? To this end, we utilized high-resolution atomic force microscopy (AFM) to map the changes in the PG organization from the pole to the cylindrical section of the rod. We extend this location-dependent imaging to interrogate different areas of the PG under different antibiotic treatment times.

To quantify the associated nanoscale directionality and orientation, we developed a MATLAB script that uses ridge detection to automatically select strands in the AFM image, quantify its orientation, and output an orientation color map and angular distribution plot. In addition, we developed an image segmentation pipeline—a combination of Otsu thresholding and particle analysis to measure gaps, holes, and breaks in the PG network. Using the aforementioned approaches, our results reveal unprecedented detail of nanometric molecular organizations with location-dependent orientation across the PG and the distribution of holes and breaks as a function of antibiotic treatment times. The increase in the frequency and size of breaks suggest the progression of death due to antibiotic-induced enzyme inactivation. Our findings reveal the remarkable application of high-resolution AFM in deciphering the bacterial cell wall; bringing us closer to the understanding of how antibiotics affect PG molecular organization and in turn, regulates its function & stability.

References

1. Vollmer, W., Blanot, D. & De Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).

2. Vollmer, W. & Bertsche, U. Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim. Biophys. Acta - Biomembr. 1778, 1714–1734 (2008).