Cloning and characterization of transcripts showing virulence-related gene expression in Naegleria fowleri

The pathogenic LEE strain of Naegleria fowleri isolated from human or mouse brain loses pathogenicity when cultured axenically in a nutrient broth. To identify genes differentially expressed in highly virulent versus weakly virulent amoebae, a cDNA library was constructed by using mRNA from amoebae recovered from a mouse brain. Two cDNA clones were isolated by differential screening of the library. The transcript homologous to clone Nf314 was preferentially expressed in highly virulent cells, whereas the transcript homologous to clone Nf435 was preferentially expressed in weakly virulent cells. Other clones showed negligible differential hybridization, but actin transcript levels were slightly elevated in the highly virulent cells. The Nf314 cDNA has an open reading frame for a 53-kDa protein 94% similar and 19% identical over 194 amino acid residues to serine carboxypeptidase from yeast cells, barley, and wheat. Southern blot analysis is consistent with a single copy of the Nf314 gene in the genome. Interestingly, the increased Nf314 transcript levels were present in cells fed on mouse brain, liver, or NIH 3T3 fibroblasts but not in cells fed on bacteria or in axenic culture. Thus, the inducer of the increased gene expression correlates with use of mammalian cells as a food source without regard to level of virulence. Since amoebae fed in culture on dissociated mouse brain were weakly virulent, as measured by their abilities to kill mice, the Nf314 gene may be required but is not sufficient for increased virulence.