Ultrastructural characterization of the stages of spheroplast preparation of Borrelia burgdorferi

Spheroplasts were generated from Borrelia burgdorferi by application of Tris-buffered lysozyme and EDTA. Cultures were examined during preparation by scanning electron microscopy (SEM) and by negative staining and/or thin sectioning followed by transmission electron microscopy (TEM). Spirochete and spheroplast proteins were also compared by electrophoresis to determine the effect of outer membrane manipulation during spheroplast preparation on the amounts of two outer surface proteins, OspA and OspB. Periplasmic flagellar release from the confines of the outer membrane began at the earliest steps of the preparation process and increased with continued progress toward the spheroplast condition. With the addition of Tris alone, spirochete uncoiling and outer membrane disruption became apparent. Lysozyme addition resulted in the formation of electron-transparent blebs where the outer membrane separated from the cytoplasmic membrane. Despite the permeability of the outer membrane to the lysozyme and the apparent alterations in its structure, OspA and B showed no decrease in concentration throughout the spheroplast preparation process. It is suggested that the flagellar release that occurs during spheroplast production also occurs in the host during Lyme disease, accounting for early immune responses to flagellin.