Dataset Information

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

ABSTRACT: This study describes an attempt to replicate experiments from the paper "Effect of BMAP-28 Antimicrobial Peptides on Leishmania major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival," which was submitted to the Reproducibility Initiative for independent validation. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) and its isomers were previously shown to have potent antiparasitic activity against Leishmania major. We tested the effectiveness of L-BMAP-28 and two of its isomers, the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), in both unamidated and amidated forms, as anti-leishmanial agents against Leishmania major promastigotes in vitro. We observed that L-BMAP-28, as well as its D and RI isomers, demonstrate anti-leishmanial activity against L. major promastigotes in vitro. The inhibitory effect was lower than what was seen in the original study. At 2 µM of amidated peptides, the viability was 94%, 36%, and 66% with L-, D- and RI-peptides, versus 57%, 6%, and 18% in the original study.

SUBMITTER: Iorns E

PROVIDER: S-EPMC4269415 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Iorns Elizabeth E Gunn William W Erath Jessey J Rodriguez Ana A Zhou Jian J Benzinou Michael M

PloS one 20141217 12

This study describes an attempt to replicate experiments from the paper "Effect of BMAP-28 Antimicrobial Peptides on Leishmania major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival," which was submitted to the Reproducibility Initiative for independent validation. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) and its isomers were previously shown to have potent antiparasitic activity against Leishmania major. We tested the effectiveness of L- ...[more]

PMID: 25517992

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Project description:BackgroundProtozoan 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 Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies.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 findingsAn 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/significanceInterestingly, 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.

Project description:In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-?, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-? against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.

Project description:BackgroundPSA (promastigote surface antigen) is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established.ResultsFrom the newly available complete genome sequences of L. major, L. infantum and L. braziliensis, we have established the complete list of PSA genes, based on the conservation of specific domain architecture. The latter includes an array of leucine rich repeats of unique signature flanked by conserved cysteine-rich domains. All PSA genes code either for secreted or membrane-anchored surface proteins. Besides the few previously identified PSA genes, which are shown here to be part of a relatively large subclass of PSA genes located on chromosome 12, this study identifies seven other PSA subtypes. The latter, whose genes lie on chromosomes 5, 9, 21 and 31 in all three species, form single gene (two genes in one instance) subfamilies, which phylogenetically cluster as highly related orthologs. On the other hand, genes found on chromosome 12 generally show high diversification, as reflected in greater sequence divergence between species, and in an extended set of divergent paralogs. Moreover, we show that the latter genes are submitted to strong positive selection. We also provide evidence that evolution of these genes is driven by intra- and intergenic recombination, thereby modulating the number of LRRs in protein and generating chimeric genes.ConclusionPSA is a Leishmania family of membrane-bound or secreted proteins, whose main signature consists in a specific LRR sequence. All PSA genes found in the genomes of three sequenced Leishmania species unambiguously distribute into eight subfamilies of orthologs. Seven of these are evolving relatively slowly and could correspond to basic functions related to parasite/host interactions. On the opposite, the other PSA gene class, which include all so far experimentally studied PSA genes, could be involved in more specialised adaptative functions.

Dataset Information

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

Publications

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

Similar Datasets

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