Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes

Abstract

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic alpha-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

“…Within this family, granulysin is most similar to NK lysin (43% identify and 67% similarity), a porcine granule protein with antibacterial activity (16). Other SAPLIP family members include the amoebapores, antibacterial peptides that amoebas use to kill bacterial prey (9). To determine the minimum active portion of the granulysin molecule retaining antimicrobial activity, peptides were synthesized to encompass the domains of structural homology with other members of this antimicrobial protein family.…”

“…The sequences for the peptides used are as follows: 1-35, GRDYRTSLTIVQKLKKMVDKPT QRSVSNAATRVSR; 36 -70, TGRSRWRDVSRNFMRRYQSRVIQGLV AGETAQQIS; 1-20, GRDYRTSLTIVQKLKKMVDK; 31-50, TRVSRT GRSRWRDVSRNFMR; 16 -35, KMVDKPTQRSVSNAATRVSR; 46 -65, RNFMRRYQSRVIQGLVAGET; and 61-80, VAGETAQQISEDLR. Instead of constructing the 62-74 peptide, additional sequence from the 15-kDa form of granulysin was used to construct the 61-80 peptide and maintain this peptide at a similar length as the others. Amoebapore A was purified from trophozoites of E. histolytica using reverse phase HPLC as the final purification step (9). Melittin was obtained from Sigma (St. Louis, MO).…”

Trophozoites ofEntamoeba histolyticaepigenetically silenced in several genes are virulence-attenuated

“…They have antibacterial activity (Leippe et al, 1994a) and are cytotoxic to human cell lines in vitro (Leippe et al, 1994b). The primary function of the pore-forming proteins in vivo is the killing of phagocytosed bacteria, but their cytolytic potency makes them a prominent virulence factor of the amoeba.…”

“…Three amoebapore isoforms termed amoebapores A, B and C have been characterized. All of them are localized in cytoplasmic granules and show only some quantitative differences in their specific activities (Leippe et al, 1994a). Structurally, the amoebapores belong to the family of saposin-like proteins (SAPLIPs) characterized by a conserved sequence motif of six cysteine residues X th EMOP, August 2008 Parasite, 2008, 15, 266-274 involved in three disulfide bridges and which comprises approximately 80 amino acid residues (Munford et al, 1995).…”

“…Although the members of the SAPLIP family have different biological functions, they are all able to interact with lipids. Amoebapores have a substantial sequence similarity with membrane-permeabilizing molecules of mammalian lymphocytes such as porcine NK-lysin and human granulysin (Leippe et al, 1994a;Andersson et al, 1995;Pena et al, 1997;Bruhn et al, 2003).…”