NACM, a cytopathogen from Naegleria ameba: Purification, production of monoclonal antibody, and immunoreactive material in NACM-treated vertebrate cell cultures

Abstract

An amebic component NACM (Naegleria ameba cytopathogenic material) is obtained from free-living amebae and acts as a cytopathogen in cultured avian and mammalian cells. NACM has been purified and partially characterized using a scheme that includes ammonium sulfate precipitation, liquid chromatographic separations and electrophoretic isolations. The purified NACM product is biologically active. Its properties, revealed by its behavior following treatment with enzymes and during purification, are those of a protein; its size is in the molecular weight range of 36000 and it has an isoelectric point of pH4.2. Monoclonal antibodies have been produced to NACM that prevent its cytopathic activity, and, in dot-blot procedures, identify purified NACM-containing fractions. When used as immunostains, NACM antibodies disclose the presence of immunospecific material both in the amebae and in vertebrate cells inoculated with NACM. In the amebae, the immunostain is located at the tips of the pseudopodia and in the peripheral cytoplasm; in the vertebrate cells, it is absent in uninoculated cells and is not observed in the inoculated cells for the first 3–4 days after the addition of the NACM to the culture. Subsequently, in a time course associated with the development of cytopathic changes observed in the light microscope, immunostaining material develops in the perinuclear cytoplasm where it condenses into a large mass before the cells undergo lysis. On the basis of these results, it appears that NACM represents an unprecedented system in which a protein material from an organism, in the course of destroying unrelated cells, causes the production of a cytoplasmic product that is immunologically recognizable as the inoculating amebic material.