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 Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies.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

Analyses of effect of BMAP-28 peptides on L. major cells.(A) Wild-type cells were (i) untreated or treated with (ii) L-BMAP-28, (iii) RI-BMAP-28 or (iv) D-BMAP-28 at 0.5 µM concentrations for 4 hours prior to fixation for TEM analyses. The figure shows cross-sections of cells at ×20,000. Notice that the untreated cell has an intact membrane, an intact cytosol and a few small vacuoles. The treated cells however possess disrupted membranes, large fluid filled vacuoles and loss of cytosolic contents; indicative of osmotic lysis. (B) Quantitative analyses of intact (clear) versus damaged (filled) cells were carried out on wt cells in presence/absence of treatment to assess the extent of the damage. Damage was assessed based on three criterion, membrane disruption, loss of cytosolic contents and presence of large fluid filled vacuoles, 120 cells were analysed by TEM for each condition.
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pntd-0001141-g002: Analyses of effect of BMAP-28 peptides on L. major cells.(A) Wild-type cells were (i) untreated or treated with (ii) L-BMAP-28, (iii) RI-BMAP-28 or (iv) D-BMAP-28 at 0.5 µM concentrations for 4 hours prior to fixation for TEM analyses. The figure shows cross-sections of cells at ×20,000. Notice that the untreated cell has an intact membrane, an intact cytosol and a few small vacuoles. The treated cells however possess disrupted membranes, large fluid filled vacuoles and loss of cytosolic contents; indicative of osmotic lysis. (B) Quantitative analyses of intact (clear) versus damaged (filled) cells were carried out on wt cells in presence/absence of treatment to assess the extent of the damage. Damage was assessed based on three criterion, membrane disruption, loss of cytosolic contents and presence of large fluid filled vacuoles, 120 cells were analysed by TEM for each condition.

Mentions: To assess morphological changes induced by the BMAP-28 isomers, L. major wild-type promastigotes were incubated with 0.5 µM L-, RI- and D-BMAP-28 peptides and prepared for transmission electron microscopy analysis (Figure 2a, b). Cell morphologies were examined based on criterion predictive of osmotic cell lysis: disrupted membrane, loss of cytosol and presence of numerous large empty vacuoles. The results suggested that cells underwent an osmotic cell lysis and followed a similar trend to the MTS viability assay. Cell morphologies were less severe effected in L-BMAP-28 treated cells, and increasingly severe in RI- and D-BMAP-28 treated cells. B-MAP-28 treated cells showed significant cell membrane disruption, losses to cytoplasmic intensity as compared to the untreated cells and significant swelling of cytoplasmic vacuoles (see also Figure S2). This severe morphological change was likely due to osmotic cell lysis and was similar to that seen for other HDPs such as histatin-5 [12].


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)

Analyses of effect of BMAP-28 peptides on L. major cells.(A) Wild-type cells were (i) untreated or treated with (ii) L-BMAP-28, (iii) RI-BMAP-28 or (iv) D-BMAP-28 at 0.5 µM concentrations for 4 hours prior to fixation for TEM analyses. The figure shows cross-sections of cells at ×20,000. Notice that the untreated cell has an intact membrane, an intact cytosol and a few small vacuoles. The treated cells however possess disrupted membranes, large fluid filled vacuoles and loss of cytosolic contents; indicative of osmotic lysis. (B) Quantitative analyses of intact (clear) versus damaged (filled) cells were carried out on wt cells in presence/absence of treatment to assess the extent of the damage. Damage was assessed based on three criterion, membrane disruption, loss of cytosolic contents and presence of large fluid filled vacuoles, 120 cells were analysed by TEM for each condition.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001141-g002: Analyses of effect of BMAP-28 peptides on L. major cells.(A) Wild-type cells were (i) untreated or treated with (ii) L-BMAP-28, (iii) RI-BMAP-28 or (iv) D-BMAP-28 at 0.5 µM concentrations for 4 hours prior to fixation for TEM analyses. The figure shows cross-sections of cells at ×20,000. Notice that the untreated cell has an intact membrane, an intact cytosol and a few small vacuoles. The treated cells however possess disrupted membranes, large fluid filled vacuoles and loss of cytosolic contents; indicative of osmotic lysis. (B) Quantitative analyses of intact (clear) versus damaged (filled) cells were carried out on wt cells in presence/absence of treatment to assess the extent of the damage. Damage was assessed based on three criterion, membrane disruption, loss of cytosolic contents and presence of large fluid filled vacuoles, 120 cells were analysed by TEM for each condition.
Mentions: To assess morphological changes induced by the BMAP-28 isomers, L. major wild-type promastigotes were incubated with 0.5 µM L-, RI- and D-BMAP-28 peptides and prepared for transmission electron microscopy analysis (Figure 2a, b). Cell morphologies were examined based on criterion predictive of osmotic cell lysis: disrupted membrane, loss of cytosol and presence of numerous large empty vacuoles. The results suggested that cells underwent an osmotic cell lysis and followed a similar trend to the MTS viability assay. Cell morphologies were less severe effected in L-BMAP-28 treated cells, and increasingly severe in RI- and D-BMAP-28 treated cells. B-MAP-28 treated cells showed significant cell membrane disruption, losses to cytoplasmic intensity as compared to the untreated cells and significant swelling of cytoplasmic vacuoles (see also Figure S2). This severe morphological change was likely due to osmotic cell lysis and was similar to that seen for other HDPs such as histatin-5 [12].

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