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 Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies.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

Induction of early apoptosis events in L. major strains after incubation with D-BMAP-28.L. major wt (black bars), L. major ko (clear bars) and L. major ko+ (grey bars) were treated with 2 µM D-BMAP-28 for 0.5 hr, 1 hr, 2 hr, and 4 hr and then analyzed for caspase 3/7 activation. L. major wt cells treated with 16 µM staurosporine for the indicated period of time were used as positive control (striped bars). Each bar represents the mean of four replicates; error bar represents the standard error of the mean.
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pntd-0001141-g004: Induction of early apoptosis events in L. major strains after incubation with D-BMAP-28.L. major wt (black bars), L. major ko (clear bars) and L. major ko+ (grey bars) were treated with 2 µM D-BMAP-28 for 0.5 hr, 1 hr, 2 hr, and 4 hr and then analyzed for caspase 3/7 activation. L. major wt cells treated with 16 µM staurosporine for the indicated period of time were used as positive control (striped bars). Each bar represents the mean of four replicates; error bar represents the standard error of the mean.

Mentions: Subsequently, we investigated the caspase activity using a caspase 3/7 assay. L. major wt, ko and ko+ cells were incubated with 2 µM D-BMAP-28 for various time intervals (<4 hr) and analyzed for caspase 3/7 activation (Figure 4). All three cell lines had minimal levels of caspase 3/7 activation in comparison to the induced caspase dependent apoptotic control, staurosporine. Of these minimal caspase 3/7 activation levels, L. major ko consistently had the lowest level compared to wt and ko+. From these results it was clear that the early D-BMAP-28 induced a caspase independent cell death.


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)

Induction of early apoptosis events in L. major strains after incubation with D-BMAP-28.L. major wt (black bars), L. major ko (clear bars) and L. major ko+ (grey bars) were treated with 2 µM D-BMAP-28 for 0.5 hr, 1 hr, 2 hr, and 4 hr and then analyzed for caspase 3/7 activation. L. major wt cells treated with 16 µM staurosporine for the indicated period of time were used as positive control (striped bars). Each bar represents the mean of four replicates; error bar represents the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001141-g004: Induction of early apoptosis events in L. major strains after incubation with D-BMAP-28.L. major wt (black bars), L. major ko (clear bars) and L. major ko+ (grey bars) were treated with 2 µM D-BMAP-28 for 0.5 hr, 1 hr, 2 hr, and 4 hr and then analyzed for caspase 3/7 activation. L. major wt cells treated with 16 µM staurosporine for the indicated period of time were used as positive control (striped bars). Each bar represents the mean of four replicates; error bar represents the standard error of the mean.
Mentions: Subsequently, we investigated the caspase activity using a caspase 3/7 assay. L. major wt, ko and ko+ cells were incubated with 2 µM D-BMAP-28 for various time intervals (<4 hr) and analyzed for caspase 3/7 activation (Figure 4). All three cell lines had minimal levels of caspase 3/7 activation in comparison to the induced caspase dependent apoptotic control, staurosporine. Of these minimal caspase 3/7 activation levels, L. major ko consistently had the lowest level compared to wt and ko+. From these results it was clear that the early D-BMAP-28 induced a caspase independent cell death.

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