Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy

Abstract

The study of structures and properties of bacterial spores is important to understanding spore formation and biological responses to environmental stresses. While significant progress has been made over the years in elucidating the multilayer architecture of spores, the mechanical properties of the spore interior are not known. Here, we present a thermal atomic force microscopy (AFM) study of the nanomechanical properties of internal structures of Bacillus anthracis spores. We developed a nanosurgical sectioning method in which a stiff diamond AFM tip was used to cut an individual spore, exposing its internal structure, and a soft AFM tip was used to image and characterize the spore interior on the nanometer scale. We observed that the elastic modulus and adhesion force, including their thermal responses at elevated temperatures, varied significantly in different regions of the spore section. Our AFM images indicated that the peptidoglycan (PG) cortex of Bacillus anthracis spores consisted of rod-like nanometer-sized structures that are oriented in the direction perpendicular to the spore surface. Our findings may shed light on the spore architecture and properties. IMPORTANCEA nanosurgical AFM method was developed that can be used to probe the structure and properties of the spore interior. The previously unknown ultrastructure of the PG cortex of Bacillus anthracis spores was observed to consist of nanometer-sized rodlike structures that are oriented in the direction perpendicular to the spore surface. The variations in the nanomechanical properties of the spore section were largely correlated with its chemical composition. Different components of the spore materials showed different thermal responses at elevated temperatures. Structures and material properties of bacterial spores play an important role in protecting them against a variety of environmental stresses, such as toxic chemicals (1), radiation (2, 3), and heat (3-5). The current knowledge of spore structures has been obtained largely using transmission electron microscopy (TEM) methods in various forms (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). A mature spore usually shows a concentric multilayer structure, consisting of the core, inner membrane, germ cell wall, cortex, outer membrane, and coat assembly. For Bacillus anthracis spores, there is an exosporium layer that loosely encases the spore. The spore core, which contains mostly DNA, RNA, enzymes, and dipicolinic acid (DPA), is in a dehydrated and metabolically dormant state (21-25). The ability of bacterial spores to survive for long durations, sometimes in harsh environments, has been attributed to the immobilization of essential DNA, RNA, and enzymes by small, acid-soluble spore proteins (SASP) (28-30). It was suggested that the DPA can be intercalated with spore DNA and RNA through covalent bonds, forming a gel-like polymer matrix (26,27). It is worth mentioning that the DPA structure in the spore core is not well understood. The germ cell wall (e.g., inner cortex) and ...

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“…The spore coat surrounds an outer membrane, which encapsulates the cortex. The cortex is made of specific peptidoglycan ( Popham, 2002 ) that is assembled into rod shaped structures, located perpendicularly to the spore surface ( Li et al, 2016 ). It confers resistance to wet heat and is essential in the dormancy of the spore as well as reducing the water content of the core ( Setlow, 2006 ).…”

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“…These proteins are structural and turned out to be highly immunogenic Despite the fact that in recent years' significant progress has been made in elucidating the structure and functions of exosporium, our understanding of the composition, mechanisms of its formation, and the role of this external structure of bacterial spores remains insufficient [10,13,55,117]. The use of modern analytical methods from the arsenal of the concept of molecular microbiology of single cells (atomic force microscopy, Raman spectroscopy, genetics) in the study of these structures is likely to become the key to a more complete understanding of the morphological, nanomechanical and biochemical characteristics of exospores [15,117,118].…”

“…Being a method of force sensing, the AFM makes it possible to obtain not only an image of the surface topography and multilayer architecture of spores, but also their nanomechanical characteristics such as elasticity, viscosity, and adhesion [15,[125][126][127]. This practice makes it possible to simultaneously obtain information on the topography of the sample surface using Young's modulus (elastic modulus) and adhesion in the form of a highresolution image [119,122,128].…”

“…R. Zolock and his colleagues showed that, based on these morphological features, four closely related species of Bacillus spores can be differentiated [130]. cross section [127], and a previously unknown peptidoglycan ultrastructure of the B. anthracis spore cortex was found, consisting of rod-like structures of nanometer size oriented in the transverse direction relative to the longitudinal axis disputes [15,118].…”

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