Abstract
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.
Highlights
We purified two polypeptides that display potent pore-forming activity and that were found after their biochemical and molecular characterization to be glycoproteins each contained in a multipeptide precursor structure
This cysteine residue motif is known from other membrane-active polypeptides defined as the protein family of saposin-like proteins (SAPLIPs) [25]
The proteins mediating this activity have been presented as potential pathogenicity factors of the respective parasite, only the pore-forming peptides of E. histolytica were isolated as a single active entity from crude extracts and characterized in detail at the molecular level [22, 23, 33]
Summary
We purified two polypeptides that display potent pore-forming activity and that were found after their biochemical and molecular characterization to be glycoproteins each contained in a multipeptide precursor structure. Pore-forming material representing naegleriapore A was eluted with 260 mM NaCl. After lyophilization of the active fractions, proteins were redissolved in 0.1% TFA and subsequently subjected to reversed-phase HPLC using a C3 PRP-3 column (Hamilton) connected to a 130A separation system (Applied Biosystems) and equilibrated with 0.1% TFA.
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