Acidocalcisomes of Phytomonas françai possess distinct morphological characteristics and contain iron.
Summary (3 min read)
INTRODUCTION
- Plant trypanosomatids include those that live in the latex of various families of lacticiferous plants, in the phloem vessels of palms, coffee trees, and other plants, in the fruit sap and seeds of several plant families, and in flowers ~Catarino et al., 2001!.
- One exception is Phytomonas françai, which infects cassava ~Manihot esculenta!, a plant of economic interest, inducing the poor development of its root system and chlorosis of aerial parts ~Kitajima et al., 1986!.
- The current chemotherapy against these parasites faces many problems that include low specificity and/or high toxicity of the drugs used and drug resistance.
- This pool has a high capacity and a low affinity for Ca21 and is able to buffer external Ca21 at concentrations in the range of 0.6–0.7 mM, as occurs with vertebrate mitochondria ~Docampo & Vercesi, 1989a, 1989b!.
Cell Cultures
- Promastigotes of P. françai isolated from cassava ~Vainstein & Roitman, 1985!.
- Were grown in Warren’s medium ~Warren, 1960! at 288C supplemented with 10% ~v/v! heat inactivated fetal bovine serum.
- At 2–3 days after inoculation, cells were collected by centrifugation and washed twice in 250 mM sucrose ~for TEM preparations!.
- Or in Dulbecco’s phosphate buffered saline ~PBS!.
- Protein was determined by using the Bio-Rad Coomassie blue method.
Chemicals
- Dulbecco’s PBS, ethylene glycol-bis~b-aminoethyl ether!N,N,N ',N '-tetraacetic acid ~EGTA!, 2-hydroxyethyl-1piperazineethanesulfonic acid ~HEPES!, protease inhibitors, and nigericin were purchased from Sigma Chemical Company ~St. Louis, MO!.
- Acridine orange ~AO! was obtained from Molecular Probes ~Eugene, OR!.
- Polyclonal antibodies raised against a keyhole limpet hemocyanin-conjugated synthetic peptide corresponding to the hydrophilic loop XII ~antibody PABHK or 326! of plant V-H 1-PPase ~Zhen et al., 1997! and aminomethylenediphosphonate ~AMDP!.
- Philip Rea, University of Pennsylvania ~Philadelphia, PA!.
- All other reagents were of analytical grade.
Conventional Transmission Electron Microscopy
- Cells were washed in Dulbecco’s PBS, pH 7.2, fixed in 2.5% glutaraldehyde, 4% paraformadehyde in 0.1 M cacodylic acid buffer, postfixed in 1% OsO4 plus 0.8% ferrocyanide and 5 mM CaCl2 in 0.1 M cacodylic acid buffer for 30 min, dehydrated in acetone series, and embedded in Polibed 812 epoxide resin.
- Observation was made in a Zeiss 900 transmission electron microscope operating at 80 kV.
Imaging of Unfixed Whole Cells
- Cells were washed and resuspended in 250 mM sucrose.
- Drops were applied to 200 mesh formvar/carbon-coated copper grids, allowed to adhere for 10 min, carefully blotted dry, and observed in an energy-filtering LEO EM 912 transmission electron microscope operating at 120 kV.
- Energy-dispersive X-ray spectra were recorded from the acidocalcisomes present in whole cells adhered to formvar/ carbon-coated copper grids.
- As control, spectra were collected from regions of the cytoplasm adjacent to acidocalcisomes and from formvar/carbon film.
- Analyses were performed using an Oxford Link ISIS system attached to the microscope.
Elemental Mapping
- X-ray mappings were acquired in a LEO 912 Omega equipped with a scanning device.
- Imaging mode, spot size was 40 nm, and emission current ;10 mA. equipped with an ATW atmospheric window.
- Analyses were performed using a Link multichannel energy analyzer and Link ISIS 3.00 software ~Oxford Instruments, Wiesbaden, Germany!.
Immunofluorescence Microscopy
- Cells fixed in freshly prepared 4% formaldehyde were allowed to adhere to poly~L-lysine!-coated coverslips, permeabilized with 0.3% Triton X-100 for 3 min, and blocked with 50 mM ammonium chloride and 3% bovine serum albumin in PBS.
- Immunofluorescence was carried out using a 1:100 dilution of anti-V-H1-PPase antibody and a fluorescein isothiocyanate-coupled goat anti-rabbit IgG secondary antibody ~1:100!.
- Fluorescence images were obtained with an Olympus BX-60 fluorescence microscope fitted with a 493-nm excitation filter, a CCD camera, and an image analysis system.
Isolation of Electron-Dense Organelles
- Electron-dense organelles were isolated by the iodixanol method similar to that described before for the isolation of acidocalcisomes of Trypanosoma cruzi ~Scott & Docampo, 2000!.
- Cell pellets of Phytomonas were mixed with approximately 23 wet weight silicon carbide ~Aldrich! and ground with a pestle and mortar until cell lysis reached 90%.
- The lysate was subsequently suspended in lysis buffer and centrifuged 10 min at 580 3 g to remove the silicon carbide, unbroken cells, and debris.
- The supernatant was centrifuged for 10 min at 15,000 3 g, and the pellet obtained was suspended in 4 ml of lysis buffer with the aid of a 22-gauge needle and applied to a discontinuous gradient of iodixanol, with 4-ml steps of 24, 28, 34, 37, and 40% iodixanol diluted in lysis buffer.
- The fraction pelleted on the bottom of the tube was also used for electron microscopy preparations.
Proton Pump Activity
- Pyrophosphate-driven proton transport was assayed by measuring changes in the absorbance of acridine orange ~A493-A530! in an SLM-Aminco DW 2000 dual wavelength spectrophotometer.
- Each experiment was repeated at least three times with different cell preparations, and Figure 4, below, show representative experiments.
RESULTS
- Electron spectroscopic images showed a large number ~208 6 25!.
- In contrast to the parasites thus far studied, the electron-dense organelles of P. françai were not all spherical.
- X-ray microanalysis performed on different regions of the cytoplasm ~Fig. 1D! and on fomvar/carbon film ~Fig. 1E! did not show the calcium, iron, and zinc peaks characteristic of the electron dense organelles.
DISCUSSION
- Previous studies applying X-ray microanalysis and/or elemental mapping have reported the presence of iron-rich organelles in different trypanosomatids.
- Iron constitutes an important element, being part of many molecules involved in several biological reactions, including oxygen transport, respiration, photosynthesis, and drug detoxification.
- Therefore, as most of the iron is enclosed by the acidocalcisome membrane ~as is apparent in the iron images; Fig. 2!, the organelle could also play a role in a mechanism of iron detoxification by the parasite.
- Additionally, the detection of a V-H1PPase activity in cell fractions, especially in the fraction enriched of electron-dense organelles, indicates that in these parasites the enzyme is active.
- As the V-H1-PPase has been found in the acidocalcisomes of a variety of parasites ~Docampo & Moreno, 2001!, and is therefore considered a marker for the organelle, the results support the idea that the iron-rich/calcium-containing organelles in P. françai correspond to acidocalcisomes.
Did you find this useful? Give us your feedback
Citations
424 citations
Cites background from "Acidocalcisomes of Phytomonas franç..."
...In some Phytomonas species, the electron-dense material seems to be arranged in a concentric patter...
[...]
94 citations
72 citations
72 citations
Cites background from "Acidocalcisomes of Phytomonas franç..."
...In trypanosomatids, acidic organelles known as acidocalcisomes, which have been identified in Phytomonas françai [85], act as the major stock of the intracellular calcium, and are implicated in processes such as calcium homeostasis, osmoregulation and polyphosphate metabolism [71]....
[...]
64 citations
Cites background from "Acidocalcisomes of Phytomonas franç..."
...donovani [23], Phytomonas françai [24], and monogenetic trypanosomatids [25], as well as in other protists (see below)....
[...]
References
60 citations
"Acidocalcisomes of Phytomonas franç..." refers background or result in this paper
...…of T. brucei were shown to be inaccessible to horseradish peroxidase ~Coppens et al., 1993!, whereas loading of T. cruzi with gold-labeled transferrin ~Scott et al., 1997! or albumin ~Miranda et al., 2000! labeled the endo-/lysosomal system in the parasites but not the acidocalcisomes....
[...]
...Acidocalcisomes have been morphologically defined in routine TEM preparations as empty vacuoles with a single membrane, circular shape, and electron-dense cores associated with the inner face of their membranes ~Scott et al., 1997, 1998; Rodrigues et al., 1999a, 1999b; Miranda et al., 2000!....
[...]
...…the presence of iron, the elemental composition of these organelles was similar to that found in acidocalcisomes of T. cruzi ~Scott et al., 1997; Miranda et al., 2000!, T. brucei ~Rodrigues et al., 1999a!, and L. donovani ~Rodrigues et al., 1999b!, thus providing evidence that, from the…...
[...]
...…appear as spherical electrondense organelles with the whole matrix filled by an electrondense material, which is mainly composed of oxygen, sodium, magnesium, phosphorus, potassium, calcium, and zinc as probed by energy-dispersive X-ray microanalysis ~Scott et al., 1997; Miranda et al., 2000!....
[...]
...Accordingly, a later work where X-ray spectra were collected from reservosomes of epimastigotes that had incorporated goldlabeled albumin showed the presence of iron in these organelles ~Miranda et al., 2000!....
[...]
59 citations
"Acidocalcisomes of Phytomonas franç..." refers background in this paper
...…are important plant pathogens, causing diseases such as phloem necrosis in coffee ~Stahel, 1931; Vermeulen, 1963!, heart rot in coconut ~Parthasarathy et al., 1976! and sudden wilt in oil palm ~Dollet et al., 1977!, and are responsible for the destruction of many plantations in…...
[...]
54 citations
"Acidocalcisomes of Phytomonas franç..." refers background in this paper
...…and for this reason they were thought to represent a phagosome or storage body containing haem derived from the incomplete digestion of hemoglobin ~Vickerman & Tetley, 1977!. Herpetomonas samuelpessoai also exhibit iron-rich electron-dense organelles similar to those found in T. cyclops, where…...
[...]
48 citations
"Acidocalcisomes of Phytomonas franç..." refers background in this paper
...A Na1/H1 antiporter that may participate in Ca21 release from these organelles has also been described in Trypanosoma brucei ~Vercesi & Docampo, 1996, Vercesi et al., 1997; Rodrigues et al., 1999a! and L. donovani ~Vercesi et al. 2000!....
[...]
43 citations
Related Papers (5)
Frequently Asked Questions (17)
Q2. What is the role of a phosphatase inhibitor in the fibroblasts?
Mobilization of intracellular calcium upon attachment of Toxoplasma gondii tachyzoites to human fibroblasts is required for invasion.
Q3. What is the mechanism of Ca21 uptake by the endoplasmic reticulum?
Ca21 uptake by the endoplasmic reticulum occurs via a Ca21-ATPase and is inhibited by high concentrations of vanadate and anticalmodulin agents.
Q4. How long did the X-rays take to be collected?
X rays were collected for 300 s using a Si~Li! detector with an ultrathin window in a 0–10-keV energy range with a resolution of 10 eV/channel.
Q5. What is the role of iron in the metabolism of the parasite?
Iron constitutes an important element, being part of many molecules involved in several biological reactions, including oxygen transport, respiration, photosynthesis, and drug detoxification.
Q6. What is the role of iron in the acidocalcisome?
It is possible that iron uptake into the acidocalcisomes occurs through an iron-transport system located in the acidocalcisome membrane.
Q7. What is the role of the acidocalcisome in the parasite’s metabolism?
Many functions have been attributed to the acidocalcisome, from calcium uptake, storage, and signaling to polyphosphate storage and a potential role in the adaptation of the parasite to environmental stresses ~Docampo & Moreno, 2001!.
Q8. What is the role of Ca21 in the invasion of host cells?
In addition, Ca21 is known to be involved in the invasion of host cells by different parasites, a process that is crucial for maintaining their life cycles ~Moreno et al., 1994; Lu et al., 1997; Vieira & Moreno, 2000!.
Q9. What is the morphology of the organelles in trypanosomatids?
it is generally believed that the presence of iron in trypanosomatids is directly related to the degradation of incorporated ironcontaining nutrients and growth factors ~i.e., hemin, hemoglobin, and hematin! that are prelocalized in the newly formed endocytic compartments.
Q10. What is the morphology of the organelles in T. cruzi?
Herpetomonas samuelpessoai also exhibit iron-rich electron-dense organelles similar to those found in T. cyclops, where iron may result from the degradation of hemin, an essential component of the culture media ~Carvalho & de Souza, 1977!.
Q11. How was the TEM used to examine the presence of acidocalcisomes?
To investigate the presence of acidocalcisomes in P. françai, unfixed whole cells were air dried on formvar/ carbon-coated TEM grids and observed in an energyfiltering TEM.
Q12. What was the X-ray spectra from the acidocalcisomes?
Electron-Probe X-Ray MicroanalysisEnergy-dispersive X-ray spectra were recorded from the acidocalcisomes present in whole cells adhered to formvar/ carbon-coated copper grids.
Q13. What is the morphology of the organelles in trypanosoma cyclops?
Trypanosoma cyclops contains a prominent iron-containing vacuole only visible by light microscopy when cells are grown in the presence of hemoglobin, and for this reason they were thought to represent a phagosome or storage body containing haem derived from the incomplete digestion of hemoglobin ~Vickerman & Tetley, 1977!.
Q14. What are the common types of trypanosomatids?
Plant trypanosomatids include those that live in the latex of various families of lacticiferous plants, in the phloem vessels of palms, coffee trees, and other plants, in the fruit sap and seeds of several plant families, and in flowers ~Catarino et al., 2001!.
Q15. What is the important characteristic of the acidocalcisomes?
In P. françai, one remarkable characteristic of the acidocalcisomes is the presence of a high concentration of iron, a feature that, up to now, was unique to this parasite, except for its presence in acidocalcisomes of bloodstream forms of T. cruzi ~Correa et al., 2002!.
Q16. What is the role of the acidocalcisome in the parasite’s iron metabolism?
as most of the iron is enclosed by the acidocalcisome membrane ~as is apparent in the iron images; Fig. 2!, the organelle could also play a role in a mechanism of iron detoxification by the parasite.
Q17. What is the composition of the organelles of P. françai?
Elongated structures resembling endoplasmic reticulum with an enlarged matrix also containing an electron-dense material were frequently seen ~Fig. 3C!. X-ray microanalysis confirmed that these organelles have the same composition as the dense organelles seen in wholecell preparations ~Fig. 3D!.Immunolocalization using polyclonal antibodies raised against a plant vacuolar proton pyrophosphatase showed a strong reaction with small intracellular structures in P. françai ~Fig. 4A,B!, indicating the presence of a H1 PPase in these parasites.