Compartmental responses to acute osmotic stress in Leishmania major result in rapid loss of Na+ and Cl−

LeFurgey, Ann; Ingram, Peter; Blum, J. J.

doi:10.1016/s1095-6433(00)00319-6

Cited by 38 publications

(36 citation statements)

References 23 publications

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“…5A, B), an elemental composition similar to that described previously for acidocalcisomes of L. donovani (Rodrigues et al 1999b) and L. major (LeFurgey et al 2001). Elemental mapping showed that most of these elements were restricted to acidocalcisomes, demonstrating their important role in ion storage in these parasites (Fig.…”

Section: Leishmania Amazonensis Cultivated In Different Media Have DIsupporting

confidence: 76%

“…A Na + /H + antiporter that may participate in Ca 2+ release from these organelles has also been described in T. brucei (Vercesi and Docampo 1996;Vercesi et al 1997;Rodrigues et al 1999a) and Leishmania donovani (Rodrigues et al 1999b;Vercesi et al 2000). X-ray microanalysis in combination with transmission electron microscopy showed that these organelles contain considerable amounts of sodium, magnesium, phosphorus, potassium, calcium, and zinc (Scott et al 1997;Rodrigues et al 1999a, b;Miranda et al 2000Miranda et al , 2004LeFurgey et al 2001). Two exceptions are the bloodstream forms of T. cruzi (Correa et al 2002) and the culture forms of Phytomonas françai (Miranda et al 2004) that have been shown to contain iron-rich acidocalcisomes.…”

Section: Introductionmentioning

confidence: 97%

“…Several functions have been attributed to the acidocalcisomes, such as storage of high energy compounds, calcium and other cations, and regulation of intracellular pH and cellular osmolarity (reviewed in Moreno 1999, 2001;De Souza et al 2000, LeFurgey et al 2001. Acidocalcisomes have also been shown to have a dynamic distribution during human foreskin fibroblast invasion by Toxoplasma gondii and adopt a collar-like appearance upon initiation of infection (Drozdowicz et al 2003).…”

Section: Introductionmentioning

confidence: 99%

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Dynamics of polymorphism of acidocalcisomes in Leishmania parasites

Miranda

Docampo

Grillo

et al. 2004

Histochemistry and Cell Biology

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Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 mm diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warren's medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture con-ditions.

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Section: Leishmania Amazonensis Cultivated In Different Media Have DIsupporting

confidence: 76%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Dynamics of polymorphism of acidocalcisomes in Leishmania parasites

Miranda

Docampo

Grillo

et al. 2004

Histochemistry and Cell Biology

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“…We show in this study that Leishmania Laps were inactivated by incubation with metalion chelators and that activity is enhanced by Mn(II), Mg(II), and Co(II) at micromolar and Ni(II) at millimolar concentrations, whereas only Mn(II) and Mg(II) could re-activate the metal-depleted Lap. Although magnesium (67 nmol⅐mg Ϫ 1 ) and trace amount of zinc have been detected in the L. major cytoplasm (41), data on the cytoplasmic levels of Co(II), Mn(II), and Ni(II) are not available. 2 Analysis of the metal content of L. amazonensis Lap by ICP-AES yielded a 2.12:1 zinc-Lap subunit ratio, whereas manganese was detected at a manganese-Lap subunit ratio of 0.05:1.…”

Section: Fig 3 Expression Of Lap Mrna and Protein In Leishmaniamentioning

confidence: 99%

Cloning and Characterization of a Leucyl Aminopeptidase from Three Pathogenic Leishmania Species

Morty

Morehead

2002

Journal of Biological Chemistry

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Aminopeptidases are emerging as exciting novel drug targets and vaccine candidates in parasitic infections. In this study, we describe for the first time an aminopeptidase from three highly pathogenic Leishmania species. Intronless genes encoding a leucyl aminopeptidase (lap) were cloned from Leishmania amazonensis, Leishmania donovani, and Leishmania major, which encoded 60-kDa proteins that displayed homology to leucyl aminopeptidases from Gram-negative bacteria, plants, and mammals. The lap genes were present as a single copy in each genome, and lap mRNA was detected by reverse transcription-PCR in all life-cycle stages of L. amazonensis. Lap assembled into catalytically competent 360-kDa hexamers and demonstrated potent amidolytic activity against synthetic aminopeptidase substrates containing leucine, methionine, and cysteine residues, representing the most restricted substrate specificity of any leucyl aminopeptidase described to date. Optimal activity was observed against L-leucyl-7-amido-4-methylcoumarin (k cat /K m Ϸ 63 s ؊1 ⅐mM ؊1 ) with a pH optimum of 8.5. Leishmania Lap activity was inhibited by metal ion chelators and enhanced by divalent manganese, cobalt, and nickel cations, although only zinc was detected in the purified Lap by inductively coupled plasma atomic emission spectroscopy, indicating that zinc is the natural Lap cofactor. Activity was potently inhibited by bestatin and apstatin in a slow binding competitive fashion, with K i * values of 3 and 44 nM, respectively. Actinonin was a tight binding competitive inhibitor (K i Ϸ 1 nM), whereas arphamenine A (K i Ϸ 70 M) and Lleucinol (K i Ϸ 100 M) were non-tight binding competitive inhibitors. Lap was not secreted by Leishmania in vitro and was localized to the parasite cytosol.Protozoan parasites of the genus Leishmania cause visceral, cutaneous, and mucosal diseases in humans, collectively referred to as leishmaniasis. Leishmaniasis is prevalent in 88 countries, with 12 million people currently infected, a further 350 million at risk, and 2 million new cases reported per year.No vaccine exists, and therapies are inadequate (1). There is a pressing need for the identification of novel leishmanial virulence factors, drug targets, and vaccines to improve our understanding, prevention, and treatment of leishmaniasis.The peptidases of parasitic protozoans (for review, see Ref.2) are becoming increasingly important as virulence factors, drug targets, and vaccine candidates in parasitic infections. However, only three peptidases from Leishmania, lysosomal cysteine peptidases (3), a cell-surface metallopeptidase (4), and a cytosolic serine oligopeptidase (5) have received attention.Aminopeptidases, which catalyze the removal of N-terminal amino acid residues from peptides and proteins (6), are emerging as novel and exciting anti-parasite targets. Vaccination of sheep with Fasciola leucyl aminopeptidase (Lap) 1 elicited high anti-Lap titers that conferred protection against fascioliasis and fascioliasis-related liver damage (7). Synthetic broad-spec...

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“…A link between acidocalcisomes and osmoregulation was evidenced by the rapid hydrolysis or synthesis of acidocalcisome polyP when epimastigotes of T. cruzi were submitted to hyposmotic or hyperosmotic stress, respectively (6). A role for acidocalcisomes in the response of Leishmania major promastigotes to osmotic stress has also been proposed on the basis of their changes in sodium and chlorine content after hyposmotic stress (7). A functional link between acidocalcisomes and the contractile vacuole complex of C. reinhardtii (3) and D. discoideum (4), which is involved in water extrusion under hyposmotic stress, has also been proposed.…”

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confidence: 99%

A Functional Aquaporin Co-Localizes with the Vacuolar Proton Pyrophosphatase to Acidocalcisomes and the Contractile Vacuole Complex of Trypanosoma cruzi

Montalvetti

Rohloff

Docampo

2004

Journal of Biological Chemistry

112

122

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We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl 2 and AgNO 3 . No glycerol transport was detected. Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H ؉ -PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.

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Compartmental responses to acute osmotic stress in Leishmania major result in rapid loss of Na+ and Cl−

Cited by 38 publications

References 23 publications

Dynamics of polymorphism of acidocalcisomes in Leishmania parasites

Dynamics of polymorphism of acidocalcisomes in Leishmania parasites

Cloning and Characterization of a Leucyl Aminopeptidase from Three Pathogenic Leishmania Species

A Functional Aquaporin Co-Localizes with the Vacuolar Proton Pyrophosphatase to Acidocalcisomes and the Contractile Vacuole Complex of Trypanosoma cruzi

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