Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle.

From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of approximately 12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change has been confirmed as has the rod to sphere micronemal shape change. Intact micronemes released directly from sporozoites exclude negative stain and appear as smooth-surfaced electron transparent particles. Biochemically purified rod-shaped C. parvum micronemes are shown to be fragile organelles that inevitably undergo variable damage during isolation, storage and subsequent specimen preparation for EM study. In the absence of glutaraldehyde fixation, damaged micronemes allow the negative stain to enter and loose their contents and during storage undergo a rod-to-sphere shape transformation. Glutaraldehyde-fixed micronemes maintain the rod shape; intact fixed micronemes still exclude negative stain but damaged micronemes reveal a complex quasi-helical arrangement of internal protein within the rod-like micronemes. Loss of this internal organized structure appears to be responsible for the micronemal shape change. This interpretation has been advanced from mutually supportive data obtained from cryoelectron microscopy of unstained vitrified samples, conventional air-dry negative staining and cryo-negative staining. Attempts to biochemically solubilize the micronemal content by lysis and ultrasonication, and separate it from the micronemal membranes, have so far met with limited success as the internal material tends to remain as a disorganized cluster of particles upon release.