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Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

Lynn MA, Kindrachuk J, Marr AK, Jenssen H, Panté N, Elliott MR, Napper S, Hancock RE, McMaster WR - PLoS Negl Trop Dis (2011)

Bottom Line: Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania.We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

View Article: PubMed Central - PubMed

Affiliation: Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, and the Department of Medical Genetics, Vancouver, Canada.

ABSTRACT

Background: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.

Methodology/principal findings: An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.

Conclusions/significance: Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

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Related in: MedlinePlus

Leishmania cell membrane permeabilization by L-, D- and RI-BMAP-28.L. major cell lines were treated with 0.5 µM (clear symbols) or 2.0 µM (filled symbols) of each of the three BMAP-28 isomers, L-BMAP-28 (▴, △), RI-BMAP-28 (○, •) and D-BMAP-28 (□, ▪) and analyzed for membrane permeabilization using SYTOX. (A) L. major wt, (B) L. major ko, (C) L. major ko+. Incubation of cells with 0.5% Triton X-100 (filled diamonds) were used as a positive control. Three complete biological replicates were performed and the average is shown, with standard deviations less than 5% for all values.
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pntd-0001141-g003: Leishmania cell membrane permeabilization by L-, D- and RI-BMAP-28.L. major cell lines were treated with 0.5 µM (clear symbols) or 2.0 µM (filled symbols) of each of the three BMAP-28 isomers, L-BMAP-28 (▴, △), RI-BMAP-28 (○, •) and D-BMAP-28 (□, ▪) and analyzed for membrane permeabilization using SYTOX. (A) L. major wt, (B) L. major ko, (C) L. major ko+. Incubation of cells with 0.5% Triton X-100 (filled diamonds) were used as a positive control. Three complete biological replicates were performed and the average is shown, with standard deviations less than 5% for all values.

Mentions: To examine cell membrane permeability we utilized a SYTOX assay, whereby a SYTOX dye enters cells following membrane disruption and fluoresces upon interaction with DNA. Wild-type, ko and ko+ leishmanial lines were incubated with 0.5 µM and 2.0 µM of L-, D- and RI-BMAP-28 for 150 minutes in the presence of SYTOX (Figure 3). All three cell lines treated with 0.5% Triton X-100 had an immediate rapid and sustained influx of SYTOX. All BMAP-28 analogues demonstrated rapid membrane permeabilization of the wt cell line at 2 µM; however, similar permeabilization at 0.5 µM was retained only for D-BMAP-28. In contrast, removal of the GP63 protease (ko cell line) resulted in the highest fluorescence of the SYTOX dye, following incubation of the cells with 2 µM L-BMAP-28 (Figure 3b). Reconstitution of GP63 (ko+) however, resulted in HDP-mediated membrane permeabilization phenotype similar to those found for the wt cells (Figure 3c).


Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

Lynn MA, Kindrachuk J, Marr AK, Jenssen H, Panté N, Elliott MR, Napper S, Hancock RE, McMaster WR - PLoS Negl Trop Dis (2011)

Leishmania cell membrane permeabilization by L-, D- and RI-BMAP-28.L. major cell lines were treated with 0.5 µM (clear symbols) or 2.0 µM (filled symbols) of each of the three BMAP-28 isomers, L-BMAP-28 (▴, △), RI-BMAP-28 (○, •) and D-BMAP-28 (□, ▪) and analyzed for membrane permeabilization using SYTOX. (A) L. major wt, (B) L. major ko, (C) L. major ko+. Incubation of cells with 0.5% Triton X-100 (filled diamonds) were used as a positive control. Three complete biological replicates were performed and the average is shown, with standard deviations less than 5% for all values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104953&req=5

pntd-0001141-g003: Leishmania cell membrane permeabilization by L-, D- and RI-BMAP-28.L. major cell lines were treated with 0.5 µM (clear symbols) or 2.0 µM (filled symbols) of each of the three BMAP-28 isomers, L-BMAP-28 (▴, △), RI-BMAP-28 (○, •) and D-BMAP-28 (□, ▪) and analyzed for membrane permeabilization using SYTOX. (A) L. major wt, (B) L. major ko, (C) L. major ko+. Incubation of cells with 0.5% Triton X-100 (filled diamonds) were used as a positive control. Three complete biological replicates were performed and the average is shown, with standard deviations less than 5% for all values.
Mentions: To examine cell membrane permeability we utilized a SYTOX assay, whereby a SYTOX dye enters cells following membrane disruption and fluoresces upon interaction with DNA. Wild-type, ko and ko+ leishmanial lines were incubated with 0.5 µM and 2.0 µM of L-, D- and RI-BMAP-28 for 150 minutes in the presence of SYTOX (Figure 3). All three cell lines treated with 0.5% Triton X-100 had an immediate rapid and sustained influx of SYTOX. All BMAP-28 analogues demonstrated rapid membrane permeabilization of the wt cell line at 2 µM; however, similar permeabilization at 0.5 µM was retained only for D-BMAP-28. In contrast, removal of the GP63 protease (ko cell line) resulted in the highest fluorescence of the SYTOX dye, following incubation of the cells with 2 µM L-BMAP-28 (Figure 3b). Reconstitution of GP63 (ko+) however, resulted in HDP-mediated membrane permeabilization phenotype similar to those found for the wt cells (Figure 3c).

Bottom Line: Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania.We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

View Article: PubMed Central - PubMed

Affiliation: Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, and the Department of Medical Genetics, Vancouver, Canada.

ABSTRACT

Background: Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.

Methodology/principal findings: An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.

Conclusions/significance: Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Show MeSH
Related in: MedlinePlus