Effects of L-Alanine and Inosine Germinants on the Elasticity of Bacillus anthracis Spores

The surface of dormant Bacillus anthracis spores consists of a multilayer of protein coats and a thick peptidoglycan layer that allow the cells to resist chemical and environmental insults. During germination, the spore coat is degraded, making the spore susceptible to chemical inactivation by antisporal agents as well as to mechanical inactivation by high-pressure or mechanical abrasion processes. While chemical changes during germination, especially the release of the germination marker, dipicolinic acid (DPA), have been extensively studied, there is as yet no investigation of the corresponding changes in the mechanical properties of the spore. In this work, we use atomic force microscopy (A FM) to characterize the mechanical properties of the surface of Bacillus anthracis spores during germination. The Hertz model of continuum mechanics of contact was used to evaluate the Young's moduli of the spores before and after germination by applying the model to load-indentation curves. The highest modulus was observed for dormant spores, with average elasticity values of 197 +/- 81 MPa. The elasticity decreased significantly after incubation of the spores with the germinants L-alanine or inosine (47.5 +/- 41.7 and 35.4 +/- 15.8 M Pa, respectively). Exposure of B. anthracis spores to a mixture of both germinants resulted in a synergistic effect with even lower elasticity, with a Young's modulus of 23.5 +/- 14.8 M Pa. The elasticity of the vegetative B. anthracis cells was nearly 15 times lower than that of the dormant spores (12.4 +/- 6.3 MPa vs 197.0 +/- 80.5 MPa, respectively). Indeed from a mechanical strength point of view, the germinated spores were closer to the vegetative cells than to the dormant spores. Further, the decrease in the elasticity of the cells was accompanied by increasing AFM tip indentation depths on the cell surfaces. Indentation depths of up to 246.2 nm were observed for vegetative B. anthracis compared to 20.5 nm for the dormant spores. These results provide quantitative information on how the mechanical properties of the cell wall change during germination, which may explain how spores become susceptible to inactivation processes based on mechanical forces during germination and outgrowth. The study of spore elasticity may be a valuable tool in the design of improved antisporal treatments.