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 Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies.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

The effect of BMAP-28 isomers on intramacrophage L. major infection.(A) The figure displays images that were collected from peritoneal macrophages infected with L. major Seidman wt strains for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 µM for an additional 24 h. Infected macrophages were stained with DAPI and examined under UV light with an upright fluorescent microscope, using 100 magnification. (B) Peritoneal macrophages were infection with L. major Seidman wt (filled) and ko strains (open) for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 and/or 2 µM for 48 h. Infections were stained with DAPI and parasite burden was quantified as an average of 100 macrophages, and expressed as a percentage of the control infection. The average number of three complete biological replicates as well as the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where * p<0.05, **p<0.005, ***p<0.0005.
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pntd-0001141-g006: The effect of BMAP-28 isomers on intramacrophage L. major infection.(A) The figure displays images that were collected from peritoneal macrophages infected with L. major Seidman wt strains for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 µM for an additional 24 h. Infected macrophages were stained with DAPI and examined under UV light with an upright fluorescent microscope, using 100 magnification. (B) Peritoneal macrophages were infection with L. major Seidman wt (filled) and ko strains (open) for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 and/or 2 µM for 48 h. Infections were stained with DAPI and parasite burden was quantified as an average of 100 macrophages, and expressed as a percentage of the control infection. The average number of three complete biological replicates as well as the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where * p<0.05, **p<0.005, ***p<0.0005.

Mentions: In the course of these studies the BMAP-28 peptides demonstrated anti-parasitic activity against the promastigote stage of the parasite, however, activity against the morphological and biochemically differentiated amastigote form has not been shown. As the amastigote stage is the disease-causing stage of the parasite, it is essential for further therapeutic development that the peptides are also active against the amastigote stage. To assess the anti-amastigote activity of the peptides an assay was created where peritoneal-derived mouse macrophages were infected with the L. major wt, ko and ko+ strains and subsequently treated with the BMAP-28 peptides at 0.5 and 2 µM concentrations. Following treatment with 0.5 µM peptide, L. major wt amastigote burdens in the infected macrophages were inhibited by 35%, 34% and 32% for L-, D- and RI-BMAP-28, respectively. Treatment with 2 µM concentrations of peptides further reduced amastigote burden by 82%, 80% and 67% for L-, RI- and D-BMAP-28, respectively. These data demonstrated that the amastigotes were killed upon the addition of the BMAP-28 isomers in a concentration dependent manner (Figure 6a and b).


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)

The effect of BMAP-28 isomers on intramacrophage L. major infection.(A) The figure displays images that were collected from peritoneal macrophages infected with L. major Seidman wt strains for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 µM for an additional 24 h. Infected macrophages were stained with DAPI and examined under UV light with an upright fluorescent microscope, using 100 magnification. (B) Peritoneal macrophages were infection with L. major Seidman wt (filled) and ko strains (open) for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 and/or 2 µM for 48 h. Infections were stained with DAPI and parasite burden was quantified as an average of 100 macrophages, and expressed as a percentage of the control infection. The average number of three complete biological replicates as well as the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where * p<0.05, **p<0.005, ***p<0.0005.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001141-g006: The effect of BMAP-28 isomers on intramacrophage L. major infection.(A) The figure displays images that were collected from peritoneal macrophages infected with L. major Seidman wt strains for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 µM for an additional 24 h. Infected macrophages were stained with DAPI and examined under UV light with an upright fluorescent microscope, using 100 magnification. (B) Peritoneal macrophages were infection with L. major Seidman wt (filled) and ko strains (open) for 24 h, and treated with L-, RI- and D-BMAP-28 at 0.5 and/or 2 µM for 48 h. Infections were stained with DAPI and parasite burden was quantified as an average of 100 macrophages, and expressed as a percentage of the control infection. The average number of three complete biological replicates as well as the standard errors are shown. Paired T-tests untreated vs treated indicated significance, where * p<0.05, **p<0.005, ***p<0.0005.
Mentions: In the course of these studies the BMAP-28 peptides demonstrated anti-parasitic activity against the promastigote stage of the parasite, however, activity against the morphological and biochemically differentiated amastigote form has not been shown. As the amastigote stage is the disease-causing stage of the parasite, it is essential for further therapeutic development that the peptides are also active against the amastigote stage. To assess the anti-amastigote activity of the peptides an assay was created where peritoneal-derived mouse macrophages were infected with the L. major wt, ko and ko+ strains and subsequently treated with the BMAP-28 peptides at 0.5 and 2 µM concentrations. Following treatment with 0.5 µM peptide, L. major wt amastigote burdens in the infected macrophages were inhibited by 35%, 34% and 32% for L-, D- and RI-BMAP-28, respectively. Treatment with 2 µM concentrations of peptides further reduced amastigote burden by 82%, 80% and 67% for L-, RI- and D-BMAP-28, respectively. These data demonstrated that the amastigotes were killed upon the addition of the BMAP-28 isomers in a concentration dependent manner (Figure 6a and b).

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