Abstract
Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals. In free-living protozoa, lysozyme-like proteins are involved in the digestion of phagocytosed bacteria. Here, we purified a protein with lysozyme activity from Dictyostelium amoebae, which constitutes the founding member, a novel class of lysozymes. By tagging the protein with green fluorescent protein or the Myc epitope, a new type of lysozyme-containing vesicle was identified that was devoid of other known lysosomal enzymes. The most highly expressed isoform, encoded by the alyA gene, was knocked out by homologous recombination. The mutant cells had greatly reduced enzymatic activity and grew inefficiently when bacteria were the sole food source. Over time the mutant gained the ability to internalize bacteria more efficiently, so that the defect in digestion was compensated by increased uptake of food particles.
Highlights
Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals
Interference with Rabs [6, 7] and LYST proteins [8, 9] produces phenotypes in Dictyostelium that are comparable with those observed in mammalian cells, indicating that many lysosomal constituents are functionally conserved in evolution
Purification of a Novel Lysozyme and Identification of the alyA Gene—We purified a protein with lysozyme activity from axenically cultured Dictyostelium amoebae, exploiting its adsorption to a Bio-Gel matrix
Summary
Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals. PCR analysis using a primer pair that binds to both alyA and alyB genes (see below) revealed both genes in genomic DNA from wild-type cells, whereas only a product corresponding to alyB was detected in the mutant (Fig. 3A).
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