Abstract
To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.
Highlights
Certain spirochete genospecies belonging to the Borrelia burgdorferi sensu lato complex are causative agents of Lyme disease (LD) and are transmitted by hard ticks of the genus Ixodes (Barbour and Hayes, 1986)
Spherical cells are named in various ways, e.g., round bodies (RBs), spheroplast L-forms, cell wall-deficient, or cystic forms, and are described as large spherical structures with numerous flagella enclosed by an intact outer membrane (Hulínská et al, 1989, 1994; Mursic et al, 1996; Brorson et al, 2001; Miklossy et al, 2008; for reviews see Stricker and Johnson, 2011; Berndtson, 2013; Lantos et al, 2014)
We used a cryo-fluorescence and cryo-SEM approach for the rapid and close-to-native assessment of both the viability and morphological changes of green fluorescent protein (GFP)-expressing spirochetes exposed to physiological stress
Summary
Certain spirochete genospecies belonging to the Borrelia burgdorferi sensu lato complex are causative agents of Lyme disease (LD) and are transmitted by hard ticks of the genus Ixodes (Barbour and Hayes, 1986). Borrelia consist of a protoplasmic cell cylinder surrounded by an outer membrane and a plasma membrane with a peptidoglycan layer (Barbour and Hayes, 1986). Both membranes enclose a periplasmic space in which the flagella are located (Goldstein et al, 1994). Flagella define the flat-wave morphology of spirochetes and are responsible for their motility. In addition to the flat-wave and rod-shaped morphological forms, the existence of non-motile atypical morphologies, such as looped/ring-shaped forms or spherical forms, has been previously described (Barbour and Hayes, 1986). Nonmotile spirochetes have been visualized in vivo within the midgut of unfed Ixodes scapularis nymphs (Dunham-Ems et al, 2009)
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