Naegleriapore A pore-forming peptide

The pore-forming polypeptides of Naegleria fowleri, naegleriapores A and B, are processed from separate multipeptide precursor structures. According to their transcripts, each precursor molecule appears to contain additional naegleriapore-like polypeptides, all of which share a structural motif of six invariant cysteine residues within their amino acid sequence. To identify the putative pronaegleriapore-derived peptides at the protein level, amoebic extracts were screened for small cysteine-rich polypeptides by fluorescently labeling their cysteine residues. Three novel naegleriapore isoforms derived from the precursor molecule of naegleriapore B were identified. Two of the isoforms were purified to homogeneity and tested for their biological activity. The pore-forming activity of the novel peptides was remarkably lower than that of the originally isolated naegleriapores, but both peptides killed bacteria by permeabilizing their cytoplasmic membranes. Collectively, these results indicate that naegleriapore isoforms with antibacterial and pore-forming activity are proteolytically released from the same precursor protein, presumably to generate a phylogenetically ancient complementary antimicrobial arsenal.

The free-living amoeboflagellate and potential human pathogen Naegleria fowleri causes the often fatal disease primary amoebic meningoencephalitis. The molecular repertoire responsible for the cytolytic and tissue-destructive activity of this amoeboid protozoon is largely unknown. We isolated two pore-forming polypeptides from extracts of highly virulent trophozoites of N. fowleri by measuring their membrane-permeabilizing activity. N-terminal sequencing and subsequent molecular cloning yielded the complete primary structures and revealed that the two polypeptides are isoforms. Both polypeptides share similar structural properties with antimicrobial and cytolytic polypeptides of the protozoon Entamoeba histolytica (amoebapores) and of cytotoxic natural killer (NK) and T cells of human (granulysin) and pig (NK-lysin), all characterized by a structure of amphipathic alpha-helices and an invariant framework of cysteine residues involved in disulfide bonds. In contrast to the aforementioned proteins, the Naegleria polypeptides both are processed from large precursor molecules containing additional isoforms of substantial sequence divergence. Moreover, biochemical characterization of the isolated polypeptides in combination with mass determination showed that they are N-glycosylated and variably processed at the C terminus. The biological activity of the purified polypeptides of Naegleria was examined toward human cells and bacteria, and it was found that these factors, named naegleriapores, are active against both types of target cells, which is in good agreement with their proposed biological role as a broad-spectrum effector molecule.