(Received for publication, October 8, 1996, and in revised form, January 9, 1997)
From the Laboratory of Molecular Parasitology,
Department of Pathobiology, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61802 and the
§ Department of Microbiology and Immunology, University
of Health Sciences, Chicago Medical School,
North Chicago, Illinois 60064
Virulent and avirulent clones of Leishmania
mexicana amazonensis promastigotes or amastigotes were loaded
with the fluorescent reagent fura 2/AM to measure intracellular free
calcium ([Ca2+]i). When the cells were treated
with the calcium ionophore ionomycin in the nominal absence of
extracellular Ca2+, there was an increase of
[Ca2+]i that was further elevated by addition of
either NH4Cl, nigericin, or the vacuolar
H+-ATPase inhibitor bafilomycin A1. Similar
results were obtained when the order of additions was reversed. Taking
into account the relative importance of the ionomycin-releasable and
the ionomycin plus NH4Cl-releasable Ca2+ pools,
it is apparent that a significant amount of the Ca2+ stored
in L. mexicana amazonensis promastigotes and amastigotes is
present in an acidic compartment rich in Ca2+
(acidocalcisome). Results indicated that more releasable
Ca2+ is stored intracellularly in virulent amastigotes than
in virulent promastigotes or avirulent cells of both stages. This
higher amount of releasable Ca2+ was correlated with the
presence of Ca2+ signals in the virulent amastigotes during
invasion of macrophages. Ca2+ signals and invasion were
reduced by preloading the parasites with intracellular
Ca2+ chelators
(1,2-bis(o-aminophenoxy)ethane-N,N,N,N
-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating
analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene
encoding an organelle-type Ca2+-ATPase was cloned and
sequenced and found overexpressed in virulent amastigotes as compared
with all other forms. Together, these results demonstrate a significant
link between expression of a Ca2+-ATPase, intracellular
Ca2+ pool content and signaling, and virulence.
Protozoan parasites of the genus Leishmania are responsible for a spectrum of diseases ranging from mild cutaneous leishmaniasis to often lethal visceral forms. The parasites live as extracellular promastigotes in the digestive tract of the insect vectors and as intracellular amastigotes in the phagolysosomes of vertebrate macrophages. The amastigotes are the only forms that maintain the infection in the vertebrate host (1).
A role for Ca2+ signaling in intracellular parasites during the process of cell invasion has been postulated after the observation of an increase in cytosolic Ca2+ concentration ([Ca2+]i)1 in some parasites upon invasion (2, 3). For example, treatment of Trypanosoma cruzi trypomastigotes with intracellular Ca2+ chelators (BAPTA/AM or quin 2/AM) to prevent an increase in their cytosolic Ca2+ resulted in an inhibition of cellular invasion (2, 4), whereas treatment with the Ca2+ ionophore ionomycin to elevate [Ca2+]i in trypomastigotes significantly enhanced their infective capacity (4).
By using fluorescent Ca2+ indicators, submicromolar levels of [Ca2+]i have been detected in promastigotes of different Leishmania species (5, 6), but no studies have been reported on amastigotes. The role of the plasma membrane of promastigotes in the regulation of their [Ca2+]i has been studied recently (7, 8). Two intracellular Ca2+ pools have been described in promastigotes of different Leishmania spp. permeabilized with digitonin (9, 10). One has characteristics typical of the mitochondria in other eukaryotic cells (11), i.e. inhibition by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and ruthenium red, a high capacity and low affinity for Ca2+, and its ability to buffer Ca2+ concentrations to a range of 0.6-0.7 µM (10). The second pool has characteristics typical of the endoplasmic reticulum (11) such as inhibition by sodium orthovanadate, stimulation with ATP, a low capacity and high affinity for Ca2+, and the ability to buffer Ca2+ concentrations to a range of 0.05-0.1 µM (10). In addition to these two intracellular Ca2+ pools there is evidence for the presence of an important third Ca2+ pool, located in an acidic compartment named the acidocalcisome, in other trypanosomatids, e.g. Trypanosoma brucei (12-14), T. cruzi (15), and in Toxoplasma gondii (16). However, nothing is known about the presence of this third Ca2+ pool in either promastigote or amastigote forms of any Leishmania species. Although nigericin has been shown to increase [Ca2+]i in Leishmania donovani promastigotes in the absence of extracellular Ca2+ (5), the high concentration of the ionophore used (4 µM) could have damaged or released Ca2+ from the mitochondria, as has been demonstrated in other trypanosomatids (17).
In this study we have examined virulent and avirulent clones of Leishmania mexicana amazonensis. We have identified the presence of acidocalcisomes in these parasites. In addition, we show that the virulence of the intracellular amastigotes is linked to their intracellular Ca2+ pool content, Ca2+ signaling during invasion, and the expression of a calcium pump that was cloned and sequenced.
Two cloned populations of virulent (clone 12-D3) and avirulent (clone 250-HA) parasites were obtained as described (18) from the same isolate of L. mexicana amazonensis (RAT/BA/72/LV78) that was originally isolated in 1972 by Lainson and Shaw (see Ref. 19), passaged in Syrian golden hamsters (R. S. Bray, Imperial College Field Station, Ascot, Berks, UK), and subsequently maintained in the laboratory of one of the authors (K.-P.C.) since 1979. One population of promastigotes was maintained in culture for more than 5 years and was subpassaged 250 times before cloning (250-HA), and the other was kept in liquid N2 since 1980 and was cultured for 12 passages before cloning (12-D3). Cloning was done by the limiting dilution method of Dwyer (20). No significant differences were observed in the growth of these promastigote populations (data not shown). Amastigotes were obtained after passage of each cloned population of promastigotes into Grace's medium (21) with 20% heat-inactivated fetal calf serum, pH 5.25, at 33 °C. This medium was also used to maintain the amastigotes. Promastigotes from L. mexicana amazonensis as well as those from L. donovani (S2 strain), Leishmania tropica (LRC-L39 strain, courtesy of Dr. F. Gamarro, University of Granada, Spain), and Leishmania major (Friedlin strain, courtesy of I. Miller, University of Notre Dame) were grown in SDM-79 medium (22), pH 7.2, containing 10% fetal calf serum, at 26 °C. Crithidia desouzai (CT-IOC 109, courtesy of Dr. Maria A. de Sousa, Brazil) were grown at 26 °C in a liquid medium containing brain-heart infusion (37 g/liter) and hemin chlorohydrate (20 mg/liter dissolved in 50% triethanolamine). Two days after inoculation, cells were collected by centrifugation. All the cells were washed twice with buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, and 50 mM Hepes buffer, pH 7.2). The final concentration of cells was determined using a Neubauer chamber. The protein concentration was determined by the biuret assay (23) in the presence of 0.2% deoxycholate.
J774.A1 macrophages were cultured in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum at 35 °C in a 5% CO2 incubator. Macrophages were infected with L. mexicana amazonensis at an estimated multiplicity of 10 parasites per macrophage in complete medium (24).
The infectivity of axenic amastigotes was determined essentially as described (24). Axenic amastigotes were taken from 3- to 4-day cultures. Amastigotes were inoculated subcutaneously in the nose of Syrian golden hamsters (Mesotricetus auratus) at 5 × 106 parasite per animal. After 2 weeks, and thereafter at weekly intervals until the 8th week, hamsters were examined and the diameter of the lesions determined using a caliper. Infectivity was calculated as the mean lesion diameter.
ChemicalsATP, ionomycin, nigericin, arsenazo III, Triton
X-100, fetal calf serum, Mops, EDTA, and EGTA were purchased from
Sigma Bafilomycin A1 was obtained from
Kamiya Biomedicals, Thousand Oaks, CA. Grace's insect cell culture
medium (catalog no. 11300-043), Trizol reagent, and Taq
polymerase were from Life Technologies, Inc. SDM-79 medium (Cat. No
57453-5L) was from JRH Biosciences, Lenexa, KS. The tetraacetoxymethyl esters of fura 2 (1-{2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2-amino-5
-methylphenoxy)-ethane-N,N,N
,N
-tetraacetic acid), BAPTA (bis-)o-aminophenoxy)ethane), quin 2 (2-{[2-bis(carboxymethyl)-amino-5-methylphenoxy]-methyl}-6-methoxy-8-bis(carboxymethyl)-aminoquinoline), and half-BAPTA (N-(2-methoxyphenyl)imidoacetic acid), fura
2/AM, BAPTA/AM, quin 2/AM, and half-BAPTA/AM, respectively, were from Molecular Probes, Inc, Eugene, OR. Poly(A)Tract mRNA isolation system,
EMBL3 phage, and pGEM-T vectors were from Promega (Madison, WI). Sequenase was from United States Biochemical Corp. All other reagents were analytical grade.
Cells were harvested, washed twice at 2,000 × g for l0 min at 4 °C in buffer A, and resuspended to a final density of l09 cells/ml in this buffer. The cell suspensions were loaded for 30 min in a 30 °C water bath under agitation (120 cycles/min) with 6 µM fura 2/AM. Subsequently, the cells were washed twice with ice-cold buffer A to remove extracellular dye and kept on ice until use. For fluorescence measurements, a 50-µl aliquot of the cell suspension was diluted into 2.5 ml of buffer A (2 × l07 cells/ml final density) in a cuvette placed in a thermostated (30 °C) Hitachi F-2000 spectrofluorometer. Excitation was at 340 and 380 nm, and emission was at 510 nm. The fura 2 fluorescence in response to the intracellular calcium concentration was calibrated from the ratio of 340/380 nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm (25). [Ca2+]i was calculated by titration with different concentrations of Ca-EGTA buffers (10). Concentrations of the ionic species and complexes at equilibrium were calculated by employing an iterative computer program (10).
Digital imaging fluorescence microscopy was performed as described (2). Macrophages grown on coverslips were observed with a microscope (Nikon, Diaphot 200) equipped with a 75-watt xenon arc epi-illuminator, 12 V, 100-watt power light illuminator, and an oil immersion objective (40 ×, fluorescence; all from Nikon). Loading of J744.A1 macrophages (grown in coverslips for 2 days) was at 37 °C for 30 min in the presence of 3 µM fura 2. After loading, the cells were washed three times with Dulbecco's phosphate-buffered saline. Culture medium containing parasites (loaded or not with fura-2) were added to loaded or unloaded macrophages, and the cells were observed by bright field or fluorescence microscopy. The images were collected with a system consisting of a CCD camera (model CH250; Photometrics Ltd., Tucson, AZ), an electronic unit (model CE 200A equipped with a 50-Hz 16-bit A/D converter), and a controller board (model NU 200; both from Photometrics Ltd.). Images were acquired and evaluated by a software package (IPLab, Signal Analytics, Vienna, VA) on a Macintosh Quadra 840 AV computer (Apple Computer, Inc., Cupertino, CA). Cells were excited first at 340 nm and then at 380 nm. Point density readings were taken for each image, and a visual display of the 340/380 nm ratio was produced.
Each experiment was repeated at least three times with different cell
preparations, e.g. Figs. 3, 4, 5, 6.
Cell Permeabilization
Variations in free Ca2+ concentrations in permeabilized cells were monitored by measuring the changes in the absorbance spectrum of arsenazo III (9, 10), using the SLM Aminco DW2000 spectrophotometer at the wavelength pair 675-685 nm.
Nucleic Acid AnalysisDNA was isolated by standard
procedures (26). Total RNA was isolated with Trizol reagent following
the manufacturer's recommendations (Life Technologies, Inc.). The
polyadenylated RNA was obtained using a Poly(A)Tract mRNA isolation
system. DNA was run in 0.8% agarose gels with TBE (9.5 mM
Tris/boric acid, 2.0 mM EDTA, pH 8.0) buffer. RNA was
electrophoresed in 1% agarose gels with 2.2 M
formaldehyde, 100 mM Mops, 40 mM sodium
acetate, 5 mM EDTA, pH 8.0 (26). Oligonucleotide primers
were designed to recognize the ATP phosphorylation site and the
ATP-binding site of cationic ATPase genes (27, 28), i.e.
5CGGGATCCGTNATNTGYWSNGAYAA3
, and 5
CGGAATTCGSRTCRTTNRYNCCR3
as the
5
primer and 3
primer, respectively. PCR was performed in a PTC-100
Programmable Thermal Controller (MJ Research, Inc., Watertown, MA) at
94 °C, 1 min, 55 °C, 2 min, and 72 °C, 2 min/cycle (30 cycles)
using Taq polymerase. PCR products were cloned into the
pGEM-T vector according to the manufacturer's instructions (Promega).
The cloned PCR products were sequenced, and the deduced amino acid
sequences were compared with the data base in GenBank. Analysis of the
deduced partial amino acid sequence of those clones was done by the
TFASTA program (Genetics Computer Group Inc., Wisconsin). A 1.2-kb PCR
clone with identity to organelle-type Ca2+-ATPases was used
to screen a genomic DNA library of L. mexicana amazonensis.
The library was constructed in lambda EMBL3 phage using 9-23-kb
EcoRI fragments of genomic DNA from L. mexicana amazonensis following the manufacturer's instructions (Promega). DNA sequencing was performed by the dideoxynucleotide chain termination method of Sanger et al. (29) either manually or with a 373A DNA Automatic Sequencer (Perkin-Elmer Applied Biosystems, Foster City,
CA). Internal oligonucleotides primers were designed to complete the
DNA sequence in both directions. DNA and deduced amino acid sequence
analyses were performed using the University of Wisconsin Genetics
Computer Group software package (GCG, version 8.0). DNA sequence data
was deposited in GenBank under the accession number U70540[GenBank].
Fig.
1 shows typical growth curves of the axenic amastigotes
about 10 subpassages after transformation. Clones 12-D3 and 250-HA had
similar logarithmic growth phases, but clone 250-HA grew slowly after 3 days reaching a peak cell density lower than clone 12-D3. Doubling
times varied between 18 and 24 h. The final cell densities of
12-D3 and 250-HA amastigotes were about 108/ml and 5 × 107/ml, respectively.
Infection of J774.A1 macrophages by the two different clones of
L. mexicana amazonensis was followed by determining the
number of intracellular amastigotes. Fig. 2A
shows the increase of 12-D3 amastigotes in macrophages. In contrast,
250-HA amastigotes disappeared with time. Fig. 2B shows
that, when inoculated into hamsters, the 12-D3 amastigotes produced
lesions that increased in size with time. The same number of 250-HA
amastigotes produced no lesions.
[Ca2+]i Increase Is Essential for Invasion of Macrophages by Virulent Amastigotes
Since a role for Ca2+ in cell invasion by intracellular parasites has been postulated (2-4), we investigated the changes in [Ca2+]i in amastigotes and promastigotes during their contact with J774.A1 macrophages at the single cell level. Parasite suspensions were added to glass slides with coverslips on which confluent monolayers of J774.A1 macrophages were grown. The host-parasite interaction was observed by phase contrast and fluorescence microscopy after incubation. When attached to macrophages, fura 2-loaded 12-D3 amastigotes were seen to increase the fluorescence in their cytoplasm as detected by ratio imaging (Fig. 3B). This increase in fluorescence was transient (<1 min) and asynchronous; not all amastigotes in contact with macrophages were highly fluorescent at the same time (Fig. 3B), although about 80% of the amastigotes observed in contact with macrophages were seen to increase their fluorescence at any time during the observation period. Interestingly, in most cases, the amastigotes with elevated Ca2+ were surrounded by macrophage pseudopodia (Fig. 3A). No changes could be detected in either avirulent (250-HA) amastigotes or promastigotes treated under the same conditions.
When 12-D3 amastigotes preloaded with the intracellular Ca2+ chelator quin 2/AM (50 µM) were used to infect J744.A1 macrophages for 1 h, their invasion rate was lower than controls (Table I). To investigate if this impairment was due to a lowering of cytosolic Ca2+ level, we loaded the amastigotes with another intracellular Ca2+ chelator, BAPTA/AM, which has a similar molecular structure and a comparable affinity for Ca2+ as does quin 2/AM,but has weak fluorescence emission (30). We could then use fura 2/AM as an indicator for cytosolic Ca2+ in cells preloaded with BAPTA/AM and exposed to NH4Cl and ionomycin, compounds capable of increasing [Ca2+]i in control preparations (see below). Under these conditions, Ca2+ concentration in control cells was 34 ± 9 nM (n = 3). After exposure to NH4Cl/ionomycin for 5 min (Fig. 4B, control), the cytosolic Ca2+ concentration was 300 ± 68 nM (n = 9). No significant changes were observed in the [Ca2+]i of BAPTA/AM-loaded amastigotes after exposure to NH4Cl/ionomycin under similar conditions (Fig. 4B, BAPTA). Similar to pretreatment with quin 2/AM, pretreatment with BAPTA/AM partially impaired the ability of amastigotes to invade macrophages (Table I). As a control for the possible toxicity of the intracellular chelators, amastigotes were loaded with half-BAPTA/AM, a compound of similar structure to BAPTA/AM but with no Ca2+ chelating action (30). No chelation of intracellular Ca2+ (Fig. 4B, half-BAPTA, dashed line) or decrease in the invasion rate of amastigotes was detected using this compound (Table I).
|
In conclusion, an increase in the [Ca2+]i occurs in virulent amastigotes when they attach to macrophages, and this is required for invasion.
[Ca2+]i Increase in Macrophages Upon Contact with Virulent Promastigotes Is Not Essential for InvasionTo investigate if changes in [Ca2+]i also occurred in the host cells upon contact with Leishmania, as it has been reported to occur in the case of infections with other intracellular parasites (see Ref. 3 for review), macrophages were loaded with fura 2 as described under "Experimental Procedures," and changes in [Ca2+]i were followed by digital imaging fluorescence microscopy. About 3 min after addition of virulent promastigotes (12-D3) (Fig. 5A), but not of any other stage of Leishmania (Fig. 5B and not shown), a transient increase in [Ca2+]i was detected in the macrophages which then returned to basal levels. However, loading of the macrophages with BAPTA-AM, quin-2-AM, or half-BAPTA-AM as described before (Table I) did not change the rate of infection with either virulent or avirulent promastigotes or amastigotes (data not shown). In conclusion, a transient increase in [Ca2+]i occurs in macrophages after their exposure to virulent promastigotes, and this is not essential for invasion, and no changes are detected in the presence of amastigotes.
Intracellular Ca2+ Pool Content in L. mexicana amazonensisSince the Ca2+ content of intracellular stores exerts a profound control over cell growth, the progression of cells through the cell cycle, and Ca2+ signaling (31, 32), we investigated the intracellular Ca2+ pool content in the different forms of L. mexicana amazonensis.
Trypanosomatids, in contrast to most mammalian cells, possess a Ca2+ pool associated with an acidic compartment or acidocalcisome (12-15). The working hypothesis is that these organelles have a vacuolar-type H+-ATPase for H+ uptake, a Ca2+/H+ countertransporting ATPase for Ca2+ uptake, and a Ca2+/H+ exchanger for Ca2+ release (12-15).
To test the presence of this type of organelle in L. mexicana amazonensis, we measured the effect of different ionophores and weak bases on their [Ca2+]i (13, 15). Qualitatively similar results were obtained with promastigotes and amastigotes (see below), regardless of their virulence. Representative data shown in Fig. 6 are from 12-D3 promastigotes. NH4Cl was used to alkalinize the cells, and the [Ca2+]i was monitored by fura 2-loading (13, 15). Since the unprotonated form of a weak base is freely permeable across cell membranes, the base (NH3) initially attains the same concentration in all cellular compartments (33) raising the cytosolic pH. At the same time, the pH of intracellular acidic compartments is considerably elevated as the weak base becomes protonated and accumulates. The ensuing leakage of the protonated base (NH4+) out of the compartments down a large concentration gradient opposes the action of the proton transporting ATPase (33). A steady state is attained when the rate of leakage of the protonated weak base balances the rate at which protons are pumped into the vesicle. Such protonophoric weak base effects on acidic compartments are well established for endocytic compartments (33). Fig. 6B shows that the addition of NH4Cl to L. mexicana amazonensis promastigotes caused an increase in [Ca2+]i, which was dose-dependent (not shown). The effect was not due to changes in osmotic pressure since no change in [Ca2+]i was observed upon the addition of up to 40 mM NaCl in the absence of an isotonic correction, and ammonium was concluded to be the active ion since (NH4)2SO4 produced identical results (data not shown).
Since these results were obtained in the nominal absence of extracellular calcium (1 mM EGTA was added), they indicate Ca2+ release from an intracellular compartment. The effects of ionomycin on the NH4Cl-induced rise of [Ca2+]i was investigated to identify the cellular origin of the Ca2+ mobilized by NH4Cl (13, 15, 34, 35). The addition of ionomycin (0.26 µM) to promastigotes previously exposed to NH4Cl (20 mM) caused a second rise in [Ca2+]i (Fig. 6B). Similar results were obtained when the order of additions was reversed (Fig. 6A). This indicates the existence in these cells of an ionomycin-sensitive pool that needs pH gradient neutralization in order for ionomycin-induced Ca2+ transport to be effective (35). This is because ionomycin binds essentially no calcium below pH 7.0 (36). So, it will not carry calcium out of acidic compartments because protons would compete with calcium for binding of the ionophore at the inner face of the membrane (34).
To further demonstrate the presence of acidocalcisomes in L. mexicana amazonensis, we incubated fura 2-loaded cells in the presence of nigericin (a H+/K+ exchanger (37) (Fig. 6D)) or bafilomycin A1 (a specific inhibitor of the vacuolar H+-ATPase (38) (Fig. 6F)) in the absence of extracellular Ca2+. In all cases there was an increase in [Ca2+]i, which was further elevated by the addition of ionomycin. Similar results were observed when the order of additions was reversed (Fig. 6, C and E), although bafilomycin A1 addition after ionomycin caused a slower Ca2+ release (Fig. 6E). In agreement with these results bafilomycin A1 addition before (Fig. 6F, BAF) or after NH4Cl addition (data not shown) also caused a slower Ca2+ release.
These results indicate that L. mexicana amazonensis cells have an acidic compartment that possesses a significant amount of Ca2+ and is sensitive to H+/K+ exchangers, inhibitors of the H+-ATPase, and weak bases, i.e. acidocalcisomes (12-16). Further characterization of these organelles is underway in our laboratory.
The steady-state basal [Ca2+]i of the Leishmania parasites in the presence of 1 mM EGTA was not significantly different between different stages with the exception of a slightly higher concentration in HA amastigotes (amastigotes 12-D3, 34 ± 9 nM (n = 10); amastigotes 250-HA, 53 ± 5 nM (n = 10); promastigotes 12-D3, 36 ± 4 nM (n = 10), promastigotes 250-HA, 32 ± 7 nM (n = 10)). However, the amount of releasable Ca2+ was at least 3-fold higher in virulent amastigotes (12-D3) as compared with all the other forms (Fig. 4A shows only a comparison between amastigotes and promastigotes of the 12-D3 clone, for clarity), and after addition of ionomycin/NH4Cl the [Ca2+]i of virulent amastigotes reached values of about 300 ± 68 nM (n = 9) or about 10-fold greater than basal steady-state values, whereas the values obtained with the rest were much lower although slightly higher in avirulent amastigotes than in promastigotes (amastigotes 250-HA, 102 ± 22 nM (n = 10); promastigotes 12-D3, 69 ± 15 nM (n = 8); promastigotes 250-HA, 41 ± 4 nM (n = 8)).
To learn whether there was a correlation between the Ca2+ content of the virulent amastigotes and their infectivity, we depleted their intracellular Ca2+ stores as indicated before (Fig. 4) and measured their infectivity to J774.A1 macrophages; the cells were treated with 1 µM ionomycin and 20 mM NH4Cl for 10 min at room temperature in buffer A containing 1 mM EGTA, washed twice, and incubated in the same buffer for 30 min in the presence of J774.A1 macrophages as described in Table I. The number of amastigotes per 100 macrophages decreased by 45% (50 ± 13.4 amastigotes/100 macrophages) as compared with untreated controls (91 ± 29 amastigotes/100 macrophages; mean ± S.D. from three different experiments). Although treated cells were viable at the end of the 10-min treatment, as judged by the trypan blue staining, when the parasites were left in the Ca2+-depleting buffer for 20 min or more they started to lyse thus indicating a toxic effect of prolonged treatment of the parasites with that buffer.
Cloning of an Organelle-type Ca2+-ATPase GeneThe higher amount of releasable Ca2+ in the virulent amastigotes seen (Figs. 3, 4) could be due to a more active Ca2+ uptake, a reduced Ca2+ leak from intracellular stores under nonstimulating conditions, or a greater storage volume. An enhanced uptake of Ca2+ seems likely since this could be mediated by the expression of an endoplasmic reticulum calcium pump that has been linked to the control of cell growth (39). We therefore investigated the expression of organelle-type Ca2+-ATPases in these parasites.
Ca2+-ATPases are members of a family of P-type ion pumps that contain conserved domains. Degenerated oligonucleotides corresponding to two of these domains, a phosphorylation site and a site involved in ATP binding (27, 28), were used to PCR-amplify specific sequences from L. mexicana amazonensis genomic DNA. The PCR products were cloned and sequenced. Analysis of the deduced partial amino acid sequence of these clones revealed that a ~1.2-kb PCR clone had the best score of sequence identity (55-65%) with the organelle-type Ca2+ATPases (or sarcoplasmic-endoplasmic reticulum (SERCA) Ca2+-ATPases), such as those from T. brucei (40), Plasmodium falciparum (41), and rabbit (42). This clone also hybridized to restriction enzyme-digested T. cruzi and T. brucei genomic DNA on Southern blots under low stringency conditions.2 Therefore, this clone was most likely encoding an organellar type Ca2+-ATPase.
To obtain the complete gene, this PCR clone was used as a probe to
screen a lambda EMBL3 genomic library of L. mexicana
amazonensis. Southern hybridization of EcoRI-digested
genomic DNA with the 1.2-kb clone revealed a single ~20-kb
hybridization band. Seventeen positive clones were obtained. Sequencing
of these clones revealed a complete open reading frame
(lmaa1, Fig. 7) with 3093 nucleotides. The
DNA sequence of the 1.2-kb PCR product was identical to the corresponding region of the gene obtained from the lambda EMBL3 genomic
library except for one nucleotide. This was apparently due to the use
of degenerate primers for the PCR. According to the initiation codon
ATG predicted (Fig. 7), the open reading frame codes for a protein of
1031 amino acids with a calculated molecular mass of 113.03 kDa.
Structure of the Coding Region and Genomic Organization of lmaa1
Analysis of the Lmaa1 amino acid sequence (Fig. 7) showed that this gene product contains all the conserved subdomains and invariant residues found in other P-type ATPases, such as the phosphorylation and ATP-binding domains (27, 28). Hydropathy analysis of the deduced amino acid sequence (not shown) revealed a profile very similar to those of SERCA pumps containing 10 transmembrane domains. A TFASTA search of protein data bases showed that Lmaa1 was most closely related to the SERCA-type Ca2+-ATPases, with 64.75% identity (76.46% similarity) over 1015 amino acids to the one from T. brucei and with ~43-~49% overall identity to those from other species, ranging from protozoa to humans; it also shared ~30-~35% identity with plasma membrane type Ca2+-ATPases and ~23-~25% identity with Na+,K+-ATPases from different species.
Other important features conserved in all known P-type ATPases are present in Lmaa1. Lmaa1 contains all the residues (Glu324, Glu777, Asn802, Thr805, Asp806, and Glu908) that were previously identified in SERCA Ca2+-ATPases as the high affinity Ca2+-binding sites in the center of the putative transmembrane domains M4, M5, M6, and M8 (Fig. 7) (43). Lmaa1 lacks the conserved amino acid sequence associated with calmodulin binding found near the C terminus of all mammalian plasma membrane Ca2+-ATPase (PMCA) isoforms (44), consistent with the Ca2+-ATPases from other lower eukaryotic organisms, i.e. TBA1 of T. brucei (45), tca1 of T. cruzi,2 PMC1P of Saccharomyces cerevisiae (46), pat1 of Dictyostelium discoideum, (47) and three gene products of Plasmodium falciparum (41, 48, 49).
The amino acid sequence Lys-Asp-Asp-Lys-Pro-Val402, which was found to be critical for the functional association of the Ca2+-ATPase of cardiac sarcoplasmic reticulum with phospholamban (50), is absent in Lmaa1. Interestingly, the residues located in transmembrane segment 3 important for thapsigargin binding to SERCA Ca2+-ATPases (51) are different in Lmaa1 (11 out of 20 residues in segment 3 are different as compared with SERCA pumps). In agreement with these results, we were unable to detect any significant increase in [Ca2+]i in fura 2-loaded D3 amastigotes in the presence of low concentrations of thapsigargin (0.1-1 µM), a known inhibitor of SERCA pumps (52).
Genomic DNA was digested with several restriction enzymes selected to
demonstrate genome copy number and hybridized at high stringency with a
1375-bp PstI fragment of lmaa1 (see Fig.
8C). Each of the seven different restriction
enzymes used produced a single band that varied in size (Fig.
8A). This suggests that lmaa1 is present as a
single copy gene in L. mexicana amazonensis. To further
confirm this, additional enzymes were selected to cut once both outside
(double digestion with BamHI and EcoRI and single digestion with XbaI; lanes 8 and 9,
respectively) and inside (PstI, lane 10) the
predicted coding region of lmaa1. The digests were probed
with a 4.3-kb XbaI fragment containing the complete coding region plus 0.7 kb of the upstream flanking region and 0.6 kb of the
downstream flanking region (Fig. 7C). A ~6.6-kb single band was detected in the sample digested with BamHI and
EcoRI (lane 8), and a single 4.3-kb band with
XbaI (lane 9). These fragments are consistent in
size with those expected from the DNA sequence obtained.
PstI-digested samples, showing a single ~1.4-kb band when
probed with the 1375-bp PstI fragment (lane 4),
allowed the detection of bands of ~1.4, ~3, and ~3.5 kb when
probed by the 4.3-kb XbaI fragment. These latter two bands
represent both flanking regions of the 1375-bp PstI
fragment. All these results are consistent with the conclusion that
lmaa1 is present as a single copy gene.
The lmaa1 gene is also present in other Leishmania spp. (Fig. 8B, lanes 11-13). Southern blot analysis of PstI-digested genomic DNA from L. donovani (lane 11), L. tropica (lane 12), and L. major (lane 13) was carried out at high stringency using the 4.3-kb XbaI fragment as a probe. Strong hybridization was obtained for all three species. The same probe did not hybridize with the DNA from C. desouzai under high stringency conditions.
Overexpression of lmaa1 in Virulent AmastigotesNorthern blot
analysis showed a single ~3.5-kb transcript in the two life cycle
stages of both virulent and avirulent L. mexicana amazonensis (Fig. 9A). The expression of
this transcript is up-regulated by 2-4-fold in the 12-D3 amastigotes
based on the intensity of the 3.5-kb bands. All samples were equally
loaded, as seen by hybridization with a full-length -tubulin gene
(53) (Fig. 9B). An increased expression of this gene could,
at least in part, explain the elevated amount of releasable
Ca2+ seen with virulent amastigotes (Fig. 4A)
and the Ca2+ signaling during their invasion of macrophages
(Fig. 3).
Permeabilization Experiments with L. mexicana amazonensis Amastigotes
The use of digitonin to permeabilize the plasma
membrane of different trypanosomatids has allowed the identification of
the mechanisms involved in Ca2+ transport in
acidocalcisomes (12-15). The results were consistent with the presence
of a Ca2+/H+-ATPase system pumping
Ca2+ into these acidic vacuoles (12, 15). To find out if
that was also the case with L. mexicana amazonensis
amastigotes, we treated these cells with digitonin in the presence of
arsenazo III to follow changes in ambient Ca2+. Addition of
20 µM digitonin to either virulent or avirulent amastigotes caused an immediate Ca2+ release (Fig.
10). No Ca2+ uptake could be detected even
reducing the digitonin concentration to 5 µM (not shown)
or using 10 µg of filipin/ml as an alternative permeabilizing agent
(Fig. 10, V, Fil.) and performing the incubations in the presence of a mitochondrial substrate (2 mM
succinate) or ATP (1 mM, data not shown) (9, 10, 12-15).
Although filipin addition produced a slower Ca2+ release
than digitonin, the final set point was similar (Fig. 10). It is
interesting to note that Ca2+ release was biphasic (Fig.
10) possibly indicating release from different intracellular pools of
different susceptibilities to the detergents. In this regard, a similar
digitonin-induced Ca2+ release has been reported in
promastigotes of Leishmania braziliensis (6). This effect
could be due to a similar sterol composition of the plasma membrane and
the intracellular membranes of amastigotes of L. mexicana
amazonensis, in contrast to what occurs with either T. brucei (12) or T. cruzi (15) or promastigotes of
different Leishmania spp. (9, 10) where selective
permeabilization of the plasma membrane is possible due to its
different sterol composition as compared with that of intracellular
compartments. This effect precluded any investigation of the possible
mechanisms of Ca2+ uptake in acidocalcisomes of these
parasites. However, it was possible to confirm that the total
releasable Ca2+ of L. mexicana amazonensis
amastigotes (21.6 ± 2.3 nmol/mg protein) was 3.8-fold higher than
that present in avirulent amastigotes (5.7 ± 0.9 nmol/mg protein)
(mean ± S.D. from two different experiments).
We have shown that a significant amount of Ca2+ within L. mexicana amazonensis promastigotes and amastigotes is located in an acidic compartment, i.e. acidocalcisome (12-16), that is sensitive to H+/K+ exchangers, inhibitors of the H+-ATPase, and weak bases. These results extend previous findings of the presence of acidocalcisomes in other trypanosomatids (12-15). Several drugs effective against Leishmania (amino acid esters, 54) or other trypanosomatids (chloroquine (55) and daunomycin (56)) have been shown to accumulate in acidic compartments (54-56). The chemotherapeutic effects of these agents may involve an alteration of Ca2+ homeostasis through disruption of this Ca2+-rich acidic compartment.
Our data also show that L. mexicana amazonensis amastigotes possess more releasable Ca2+ than promastigotes. The concentration of Ca2+ in amastigote-containing phagolysosomes in macrophages is not known, but if it were similar to that in the cytosol (about 0.1 µM) it would be dramatically different from the concentration of Ca2+ to which extracellular parasites are exposed (about 1 mM). The higher amount of releasable Ca2+ in amastigotes would then indicate an adaptation to an intracellular environment. Alternatively, it has been indicated that the parasitophorous vacuoles of Leishmania constitute a continuing part of the phagosome-lysosome vacuolar apparatus open to the extracellular menstruum from which exogenous substances can be brought into contact with amastigotes via pinocytosis and/or phagocytosis of macrophages during the entire period of infection (57). If this were the case, no difference in Ca2+ would be expected to exist between the extracellular space and the phagolysosomes. The higher amount of releasable Ca2+ in amastigotes would then indicate a specific function for Ca2+ in these forms, such as in signaling during invasion.
Axenic in vitro cultivation of amastigotes has been previously reported for Leishmania mexicana (58), Leishmania pifanoi (59), Leishmania panamanensis (60), L. braziliensis (60), L. major (61), L. donovani (61, 62), and L. mexicana amazonensis (63). In this study, two clones of L. mexicana amazonensis were adapted to grow axenically as amastigotes. A clear link between their intracellular Ca2+ pool content, Ca2+ signaling during macrophage invasion, expression of a Ca2+-ATPase, and virulence could be established. Virulent amastigotes were shown to have a higher amount of releasable Ca2+, greater expression of an organelle-type Ca2+-ATPase, and to increase their [Ca2+]i upon contact with macrophages. This increase in their [Ca2+]i was shown to be important for invasion of macrophages since preincubation of the parasites with intracellular Ca2+ chelators, but not with a chemical analog unable to chelate Ca2+, was able to decrease their infection of macrophages. The organelle-type Ca2+-ATPase from L. mexicana amazonensis, the gene for which was cloned and sequenced, is developmentally regulated as indicated by its higher expression in amastigotes than in promastigotes. The location of the protein product of the lmaa1 gene is not known at this time. By comparing Lmaa1 with other P-type ATPases, the sequence data indicate that this single copy ATPase gene is more closely related to the family of organellar sarcoplasmic-endoplasmic reticulum calcium (SERCA) pumps. However, the residues located in transmembrane segment M3 that have been found to be important for thapsigargin binding and that are conserved in all SERCA Ca2+-ATPases (51) are different in Lmaa1, and this correlates with the absence of effect of low concentrations of thapsigargin on the [Ca2+]i of fura 2-loaded cells. A comparison of the sequence of this transmembrane segment M3 in Lmaa1 with those of other Ca2+-ATPases indicates that Lmaa1 shares 9 out of 20 amino acid residues with SERCA pumps and 14 out of 20 residues with T. brucei Ca2+-ATPase TBA1 (45). Interestingly, TBA1 only shares 9 out of 20 amino acid residues with SERCA pumps, and 8 of these 9 amino acid residues are in common with Lmaa1 (see Scheme 1 showing the alignment of Lmaa1 with TBA1, SERCA1, SERCA2, and SERCA3; : and · indicate identical and similar amino acid residues, respectively).
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Although the increase in Ca2+ pump mRNA does not prove that increased pump protein is responsible for the increased Ca2+ content in virulent amastigotes, it is highly suggestive of this possibility. The presence of a H+-countertransporting Ca2+-ATPase involved in Ca2+ sequestration that resides in the acidocalcisomes of other trypanosomatids such as T. brucei (12) and T. cruzi (15) has been postulated. The vanadate sensitivity of all non-mitochondrial Ca2+ uptake by permeabilized trypanosomatids (12, 15) argued against the involvement of the type of Ca2+/H+ antiporter believed to exist in the vacuoles of fungi and higher plants (64, 65). However, it has also been demonstrated (12) that, as occurs with L. mexicana amazonensis (Figs. 4 and 6), the inside pH gradient due to the vacuolar proton pump facilitates Ca2+ uptake and retention. Therefore, we cannot rule out that overexpression of a vacuolar proton pump could also occur in virulent amastigotes and be responsible in part for their higher Ca2+ content. Alternatively, a reduced H+ permeability of the acidocalcisomes of the virulent parasites could also account for part of their increased Ca2+ content. In this regard, Ca2+ release from the acidocalcisomes of T. brucei procyclic trypomastigotes via a Ca2+/nH+ antiporter has been shown to be stimulated by Na+ via a Na+/H+ antiporter (14). Further work is necessary to clarify the role of all these pumps and exchangers in Ca2+ uptake and retention by trypanosomatid acidocalcisomes. However, the high sensitivity of L. mexicana amazonensis to digitonin or filipin permeabilization (Fig. 10) precludes the use of these parasites as a model for these studies.
It has been demonstrated that the Ca2+ content of intracellular stores exerts a profound control over cell growth and the progression of cells through the cell cycle (31, 32), and that growth changes can result from the inability of Ca2+ to be pumped into the intracellular stores (32). Depletion of Ca2+ from within the intracellular stores has considerable inhibitory effect on the folding and processing of proteins (66, 67). It is clear that a range of Ca2+-binding proteins in these stores are involved in not just the storage of Ca2+ within the lumen but are also functioning as "Ca2+-dependent chaperonins" to assist the folding and correct assembly of proteins (39). An alternative or additional possibility is that the decrease in Ca2+ within intracellular stores in the avirulent amastigotes (250-HA) may preclude important Ca2+ signals. These signals may be generated through release of Ca2+ from the stores and essential for the parasites to progress through the cell cycle and maintain growth or to replicate successfully within mammalian cells. Although 250-HA amastigotes have the capacity to invade macrophages in cultures or animals, they have lost their ability to replicate intracellularly and develop a successful infection. The results provide not only further support to the postulated link between intracellular Ca2+ pool content, expression of Ca2+-ATPases, Ca2+ signaling, and cell growth in eukaryotic cells (31, 32, 39) but also evidence for an important link with virulence of intracellular parasites.