ULTRASTRUCTURAL ANALYSIS OF ANTIGENIC DETERMINANTS IN PATHOGENS

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Abstract

Lyme disease is a multi-system disorder claiming increasing medical interest worldwide. The causative agent is a highly motile, helical microorganism measuring approximately 15 micrometers long and 0.2 micrometers wide and is classified as a member of the genus Borrelia. An outer sheath completely surrounds the protoplasmic cylinder. Between this protoplasmic cylinder and the outer sheath are wound the periplasmic flagella. While this description accurately describes both laboratory cultured Borrelia and clinical samples derived from Lyme disease patients, careful microscopy often reveals the presence of other structural forms which appear to be associated with borrelial growth. These structures are of special interest and questions arise as to the role, if any, of these detectable bioproducts in the pathogenesis of Lyme disease. We have shown that naturally elaborated, outer membrane blebs are frequently seen both attached to spirochetes and free in growth media. These are produced in enormous numbers in Borrelia burgdorferi cultures and in other gram negative microorganisms as well. Using both scanning (SEM) and transmission electron microscopy (TEM), we demonstrated the presence of intact borrelial DNA molecules within these structures. Subsequently, using a capture/detection method, we were able to find evidence of these structures in infected ticks, animals and humans and, importantly, this material was shown to possess a potent, non-specific, mitogenic activity for murine B-cells. While biologically significant concentrations of several eukaryotic cell inhibitors showed little effect on the growth of Borrelia burgdorferi when added to BSK-II cultures, PPMP, a sphingolipid analogue, showed immediate and profound inhibitory effects when monitored by dark-field microscopy, TEM, field emission SEM and by optical density measurements. PPMP(dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol) is known to inhibit the synthesis of sphingomyelin in chinese hamster ovary cells, to inhibit the synthesis of glucosylceramides in a large variety of mammalian cells, and to affect the tubovesicular membrane network of malaria infected erythrocytes. No direct effect on prokaryotic cells has been demonstrated. However, PPMP concentrations as low as 225 micromolar produced detectable effects on growth of Borrelia burgdorferi. Ultrastructural examination of these cultures by both SEM and TEM revealed a striking conversion of the worm-like, spirochetal form to a spherical morphology. Intermediate forms were readily detectable and the effect appeared to be both dose and time dependent. While spheroplasting of spirochetes and many other bacteria have been reported following antibiotic treatment, this is the first description of such a morphological alteration using a specific lipid analogue. Backscattered electron detection following staining with the cancer drug, Cisplatin, has revealed DNA lining the inner surface of the outer membrane of the borrelial spheroplasts.