Plasma Membrane Oxidoreductases in Control of Animal and Plant Growth
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Iron-depleted medium was prepared similarly to regular M without addition of hemin and by replacing regular FBS with iron-depleted FBS. Promastigote growth curves were initiated with parasites taken from cultures in the stationary phase of growth and resuspended at the indicated densities in fresh growth media containing or not the supplemental metal salts. A negative control containing all reaction components except the enzyme was included and analyzed by real-time PCR to exclude the possibility of DNA contamination in the RNA samples.
The target gene expression levels were quantified according to a standard curve prepared from a ten-fold serial dilution of a quantified and linearized plasmid containing the DNA segment to be amplified. For protein level determinations, total lysates of L. All constructs were transfected by electroporation [ 51 ]. The linearized resulting constructs were transfected by electroporation in 2 independent rounds [ 51 ].
For immunofluorescence of intracellular amastigotes, coverslips with BMM infected as described below were used. Slides were mounted with ProLong Gold antifade reagent Invitrogen. The construct was introduced into Arabidopsis thaliana wild type ecotype Ler and irt mutants ecotype Wassilewskija [ 31 ] via Agrobacterium by floral dipping [ 54 ].
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The irt mutant was kindly provided by Dr. When plants were grown in soil, iron was supplied as Sequestrene Sequestrene Fe Chelate, Basf water solution 0. When plants were grown on media plates, surface sterilized seeds were germinated on half-strength Murashige and Skoog medium containing 0. Mesophyll protoplasts of transgenic plants were prepared as previously described [ 55 ].
Plasma Membrane Oxidoreductases in Control of Animal and Plant Growth
Imaging of protoplasts and whole roots of transgenic plants was carried out using a Leica SPX5 confocal microscope. At the end of the uptake period cells were washed 3 times with cold HBSS. The radioactivity in the lysates was determined by liquid scintillation counting. Inductively coupled plasma mass spectrometry ICP-MS analysis was performed as previously described [ 56 ]. Each sample was subjected to two promastigote samples or three plant samples independent injections for the ICP-MS run. Infective metacyclic forms were purified from stationary phase promastigote cultures second to third day after entering stationary phase using the 3A.
The cells were washed 3 times with PBS and fixed or incubated in BMM media to complete 24, 48, 72 and 96 h of infection. The number of intracellular parasites was determined by counting the total macrophages and the total intracellular parasites per microscopic field Nikon E epifluorescence microscope.
At least host cells, in triplicate, were analyzed for each time point. After 4 h fluorescence levels were determined using a fluorescence plate reader SpectraMax M5, Molecular Devices at nm excitation and nm emission. Lesion progression was monitored once a week by measuring the difference in thickness between the left and right hind footpads with a caliper Mitutoyo Corp. The parasite load in the infected tissue was determined after 10 weeks in the infected tissue collected from footpad lesions of sacrificed mice by limiting dilution [ 38 ].
The data meet the assumptions of the test. Variance is similar among compared groups. P values and the number of times each experiment was repeated are stated in the figure legends. No statistical method was used to predetermine sample size. All animal work was conducted in accordance with the guidelines provided by National Institutes of Health for housing and care of the laboratory animals and performed under protocol R approved by the University of Maryland College Park Institutional Animal Care and Use Committee on January 11, Green, GFP.
The targeting fragments are shown below the LIR1 chromosomal locus.
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The arrows indicate the position of oligonucleotide primers used for PCR confirmation of the insertions. The brackets below the gels indicate the pair of oligonucleotide primers used for each sample. The graph shows the individual values and means of duplicate experiments.
The values correspond to the average of triplicate determinations. AB 72 h. We thank J. Hauzel University of Maryland for excellent technical assistance and J. Kaplan, D. Ward University of Utah , H. Sze, M. Corrote and B. Mittra University of Maryland for helpful discussions. We are also grateful to P.
Beverley Washington University for their generous gift of plasmids, L. Floeter-Winter for plasmids and anti-arginase antibodies, C.
Beaven University of Maryland and the Department of Cell Biology and Molecular Genetics Imaging Core for assistance with deconvolution and confocal fluorescence microscopy. Abstract Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Author summary Leishmaniasis is a human infectious disease that is endemic in many tropical and subtropical areas of the world. Introduction Leishmania spp are intracellular protozoan parasites that cause human leishmaniasis, a disease spectrum that can vary from self-healing cutaneous lesions to lethal visceralizing disease.
Results Leishmania encodes a MFS-type plasma membrane protein that is upregulated in response to excess extracellular iron Analysis of the previously published whole-genome transcriptome profile of L. Download: PPT. Fig 1. Fig 2. LIR1 localizes at the plasma membrane of promastigotes and intracellular amastigotes. LIR1 expressed in Arabidopsis thaliana is targeted to the plasma membrane and decreases the iron content of plant tissues Given the sequence similarity of LIR1 with plant nodulin-like proteins, we investigated the molecular function of LIR1 in a heterologous system by generating transgenic Arabidopsis thaliana plants expressing LIR1.
Fig 3. LIR1 expressed in Arabidopsis thaliana is targeted to the plasma membrane and decreases the iron content of leaves. LIR1 regulates intracellular iron levels in Leishmania , exporting iron to prevent its intracellular accumulation To assess the functional role of LIR1 in L. Fig 4.
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LIR1 expression mediates iron efflux and prevents accumulation of iron and other transition metals in Leishmania. Fig 5.
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LIR1 deficiency enhances the toxic effect of excess iron and other transition metals during promastigote replication. Fig 6. LIR1 deficiency markedly reduces L. Discussion Despite advances in our understanding of pathways of iron acquisition and metabolism in Leishmania [ 13 , 14 , 23 , 39 — 42 ], how these parasites regulate their intracellular labile iron pool to prevent toxicity is still an open question.
Materials and methods Leishmania cultivation The L. Generation of L.
Plasma Membrane Oxidoreductases in Control of Animal and Plant Growth | Frederick Crane | Springer
Elemental iron quantification Inductively coupled plasma mass spectrometry ICP-MS analysis was performed as previously described [ 56 ]. Ethics statement All animal work was conducted in accordance with the guidelines provided by National Institutes of Health for housing and care of the laboratory animals and performed under protocol R approved by the University of Maryland College Park Institutional Animal Care and Use Committee on January 11, Supporting information.
S1 Table. Oligonucleotides used in this study. S1 Fig. Modulation of LIR1 transcript levels by iron availability.
S2 Fig. LIR1 expression in Arabidopsis thaliana. S3 Fig. S4 Fig. Impact of LIR1 deficiency and add-back on promastigote replication: Effect of excess iron and growth curves of independent LIR1 deficient and add-back clones. S5 Fig. Infectivity of independent LIR1 deficient and add-back clones. Acknowledgments We thank J. References 1. Shaw J. The leishmaniases—survival and expansion in a changing world. A mini-review. Mem Inst Oswaldo Cruz. Risk analysis and prediction of visceral leishmaniasis dispersion in Sao Paulo State, Brazil. Urban parasitology: visceral leishmaniasis in Brazil.
Trends Parasitol. Leishmaniases in the Mediterranean in the era of molecular epidemiology.