Dataset Information

Farnesyl pyrophosphate synthase is essential for the promastigote and amastigote stages in Leishmania major.

ABSTRACT: Isoprenoid synthesis provides a diverse class of biomolecules including sterols, dolichols, ubiquinones and prenyl groups. The enzyme farnesyl pyrophosphate synthase (FPPS) catalyzes the formation of farnesyl pyrophosphate, a key intermediate for the biosynthesis of all isoprenoids. In Leishmania, FPPS is considered the main target of nitrogen containing bisphosphonates, yet the essentiality of this enzyme remains untested. Using a facilitated knockout approach, we carried out the genetic analysis of FPPS in Leishmania major. Our data indicated that chromosomal null mutants for FPPS could only be generated in presence of an episomally expressed FPPS. Long-term retention of the episome by the chromosomal FPPS-null mutants in culture and in infected BALB/c mice suggests that FPPS is indispensable. In addition, applying negative selection pressure failed to induce the loss of ectopic FPPS in the chromosomal FPPS-null mutants, although it led to significant growth delay in culture and in mice. Together, our findings have confirmed the essentiality of FPPS in both promastigotes and amastigotes in L. major and thus validate its potential as a drug target for the treatment of cutaneous leishmaniasis.

SUBMITTER: Mukherjee S

PROVIDER: S-EPMC6529949 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Farnesyl pyrophosphate synthase is essential for the promastigote and amastigote stages in Leishmania major.

Mukherjee Sumit S Basu Somrita S Zhang Kai K

Molecular and biochemical parasitology 20190326

Isoprenoid synthesis provides a diverse class of biomolecules including sterols, dolichols, ubiquinones and prenyl groups. The enzyme farnesyl pyrophosphate synthase (FPPS) catalyzes the formation of farnesyl pyrophosphate, a key intermediate for the biosynthesis of all isoprenoids. In Leishmania, FPPS is considered the main target of nitrogen containing bisphosphonates, yet the essentiality of this enzyme remains untested. Using a facilitated knockout approach, we carried out the genetic analys ...[more]

PMID: 30926449

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 Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies.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

Dataset Information

Farnesyl pyrophosphate synthase is essential for the promastigote and amastigote stages in Leishmania major.

Publications

Farnesyl pyrophosphate synthase is essential for the promastigote and amastigote stages in Leishmania major.

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