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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 Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies.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

MTS viability assay of Leishmania major strains when treated with BMAP-28 variants.(A) L. major Friedlin strain was treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. (B) L. major Seidman strain wild-type (wt, filled bars), GP63 knockout (ko, clear bars) and reconstituted (ko+, grey bars) were treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. Cell viability was assessed at 490 nm and expressed as a percentage of untreated control cells. Three complete biological replicates were performed and the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where *p<0.05, **p<0.005, ***p<0.0005. The bar graph was created using GraphPad prism 4.
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pntd-0001141-g001: MTS viability assay of Leishmania major strains when treated with BMAP-28 variants.(A) L. major Friedlin strain was treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. (B) L. major Seidman strain wild-type (wt, filled bars), GP63 knockout (ko, clear bars) and reconstituted (ko+, grey bars) were treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. Cell viability was assessed at 490 nm and expressed as a percentage of untreated control cells. Three complete biological replicates were performed and the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where *p<0.05, **p<0.005, ***p<0.0005. The bar graph was created using GraphPad prism 4.

Mentions: To test the anti-leishmanial activities of the BMAP-28 variants, specifically the effects on the viability of promastigote L. major Friedlin, an MTS viability assay was performed. An MTS assay measures the mitochondial dehydrogenase levels of viable cells. Unmodified peptide (L-BMAP-28), the retro-inverso isoform (RI-BMAP-28) and the D- amino acid isoform (D-BMAP-28) were incubated with L. major Friedlin promastigotes in vitro (Figure 1a). Cell viability was assessed at 490 nm and expressed as a percentage of the untreated control. Cells remained 100% viable upon treatment with 0.5 µM L- and RI-BMAP-28, but experienced a 30% reduction in cell viability when treated with D-BMAP-28. Upon addition of 2 µM concentrations of the HDPs, the HDPs demonstrated a concentration dependent effect on L. major Friedlin. Cell viability was reduced to 60%, 20% and 5% when treated with L-, RI- and D-BMAP-28, respectively. Of the three BMAP-28 variant forms, the D- form of the peptide was the most potent.


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)

MTS viability assay of Leishmania major strains when treated with BMAP-28 variants.(A) L. major Friedlin strain was treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. (B) L. major Seidman strain wild-type (wt, filled bars), GP63 knockout (ko, clear bars) and reconstituted (ko+, grey bars) were treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. Cell viability was assessed at 490 nm and expressed as a percentage of untreated control cells. Three complete biological replicates were performed and the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where *p<0.05, **p<0.005, ***p<0.0005. The bar graph was created using GraphPad prism 4.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001141-g001: MTS viability assay of Leishmania major strains when treated with BMAP-28 variants.(A) L. major Friedlin strain was treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. (B) L. major Seidman strain wild-type (wt, filled bars), GP63 knockout (ko, clear bars) and reconstituted (ko+, grey bars) were treated with 0.5 µM and 2 µM concentrations of L-, RI- and D-BMAP-28 for 4 hours. Cell viability was assessed at 490 nm and expressed as a percentage of untreated control cells. Three complete biological replicates were performed and the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where *p<0.05, **p<0.005, ***p<0.0005. The bar graph was created using GraphPad prism 4.
Mentions: To test the anti-leishmanial activities of the BMAP-28 variants, specifically the effects on the viability of promastigote L. major Friedlin, an MTS viability assay was performed. An MTS assay measures the mitochondial dehydrogenase levels of viable cells. Unmodified peptide (L-BMAP-28), the retro-inverso isoform (RI-BMAP-28) and the D- amino acid isoform (D-BMAP-28) were incubated with L. major Friedlin promastigotes in vitro (Figure 1a). Cell viability was assessed at 490 nm and expressed as a percentage of the untreated control. Cells remained 100% viable upon treatment with 0.5 µM L- and RI-BMAP-28, but experienced a 30% reduction in cell viability when treated with D-BMAP-28. Upon addition of 2 µM concentrations of the HDPs, the HDPs demonstrated a concentration dependent effect on L. major Friedlin. Cell viability was reduced to 60%, 20% and 5% when treated with L-, RI- and D-BMAP-28, respectively. Of the three BMAP-28 variant forms, the D- form of the peptide was the most potent.

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