(Received for publication, October 18, 1996, and in revised form, December 31, 1996)
From the Molecular Biology Unit, Tata Institute of Fundamental Research, Homi Bhabha Road, Bombay, 400 005 India
A cDNA expression clone of the human malarial
parasite Plasmodium falciparum, Pf4, which was reactive
only to the immune sera and not to the patient sera, has recently been
found to be the P. falciparum homologue of the P0 ribosomal
phosphoprotein gene. A Northern analysis of the P0 gene revealed the
presence of two transcripts, both present in all the different
intraerythrocytic stages of the parasite life cycle. A 138-base pair
amino-terminal domain of this gene was expressed as a fusion protein
with glutathione S-transferase in Escherichia
coli. Polyclonal antibodies raised against this domain
immunoprecipitated the expected 38-kDa P0 protein from the
35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone
Pf4 also
immunoprecipitated the same size protein from
[35S]methionine-labeled P. falciparum protein
extract. Purified IgG from polyclonal antibodies raised against the
amino-terminal domain of P0 protein completely inhibited the growth of
P. falciparum in vitro. This inhibition appears to be
mainly at the step of erythrocyte invasion by the parasites.
It has been documented that people living in malaria-endemic areas
acquire immunity to Plasmodium falciparum after repeated infections. The nature of this immunity is poorly understood at the
molecular level. It is apparent from studies involving passive transfer
of IgG from immune adults to the non-immune subjects that circulating
antibodies do play an important role (1, 2). The specificity of these
protective antibodies is as yet unknown. It has been shown that the
antibodies present in immune adults recognize domains that are
conserved in different strains of P. falciparum (3).
However, it has also been documented that many malarial antigens
possess repetitive protein domains, which evoke a strong antibody
response. Many of these antibodies are non-protective, and the
corresponding malarial epitopes are postulated to be immune-evasive or
smokescreen domains (4, 5). Thus, to search for pan-specific and
possibly protective antibodies, a differential immunoscreening of an
erythrocytic stage-specific cDNA expression library of P. falciparum was carried out using malaria-immune and acute patient sera. This resulted in the identification of several novel cDNA clones, which reacted exclusively and yet extensively with immune sera
samples (6). The clone Pf4, which was reactive to the largest number
of immune sera (80 out of 92), has been cloned and sequenced recently
(7). This was found to be the P. falciparum gene homologue
of the ribosomal phosphoprotein P0
(PfP0).1
Ribosomal phosphoprotein P0 is considered to be related to the family of the acidic ribosomal phosphoproteins P1 and P2, because of the highly homologous carboxyl-terminal domain (8). Antibodies against this domain coprecipitate all three P proteins (9). P0 could be cross-linked to P1 and P2 protein in Artemia salina ribosomes (10), and these data, along with that from yeast cells (11), strongly indicate the existence of a (P1)2·P0·(P2)2 protein complex in the eukaryotic ribosomes. This complex has been compared with the bacterial complex L10·(L7/L12)2, which forms the stalk of the large subunit at the GTPase domain along with the 23 S ribosomal RNA (12, 13). It has been documented that P0 protein is absolutely required for the ribosomal activity and cell viability in yeast (14). The conserved carboxyl-terminal domain of the P proteins is very antigenic and found to be the main antigenic target for sera reactivity of about 10-15% of patients of the autoimmune disorder systemic lupus erythematosus (15). Antibodies to this domain have also been detected in patients of suffering from diseases caused by protozoan parasites such as Chagas' heart disease (16) and leishmaniasis (17). In this paper we report the characterization of this protein from P. falciparum, and we show for the first time that P0 is indeed a phosphoprotein. We also show that antibodies raised against the amino-terminal domain of this protein inhibits P. falciparum growth in vitro.
All reagents, unless otherwise specified, were
purchased from Sigma. [-32P]dATP,
[32P]orthophosphoric acid were provided by the Board of
Radiation Technologies, India. [35S]Methionine and
[35S]cysteine were purchased from Amersham International
(Buckinghamshire, England).
Asexual stages of FCR3 (Gambia) and the FCK2 (India) strains of P. falciparum were cultured in vitro at 37 °C in the presence of human erythrocytes of serological type O+ in complete medium (RPMI 1640 medium containing 28 mM NaHCO3, 25 mM HEPES, and supplemented with either 10% human serum or 0.5% Albumax (Life Technologies, Inc.) and 80 µg/ml Gentamycin sulfate) in sterile Petri dishes using the candle-jar method or sealed flasks flushed with 5% O2, 5% CO2, and 90% N2 gas mixture (18). For stage-specific RNA preparation and in vitro parasite growth inhibition assays, cultures were synchronized by sorbitol treatment according to the method of Lambros and Vanderberg (19). Intracellular parasites from each of the substages were liberated from infected erythrocytes by saponin lysis (20) for total RNA and genomic DNA extraction. The gametocytic stages of the parasite (NF54 strain) used for the immunofluorescence studies were kindly provided by Dr. Nirbhay Kumar, Johns Hopkins University (21).
Preparation of Nucleic AcidsTotal cellular RNA was extracted using a single step method described by Chomczynski and Sacchi (22). Genomic DNA was extracted from total erythrocytic stages of the parasite as described in detail elsewhere (6).
Southern and Northern HybridizationSouthern and Northern
hybridization were performed with the radioactively labeled
[-32P]dATP 251-bp
Pf4 cDNA fragment as well as
the 700-bp L-4-7 (carboxyl-terminal fragment of PfP0 protein) with
specific activity of 2 × 108 cpm by following the
membrane manufacturer's protocol (Amersham International) in the
presence of 50% formamide. Briefly, 2 µg of parasite genomic DNA cut
with appropriate restriction enzymes (New England Biolabs Inc.) was
electrophoresed in a 1.0% agarose gel. In the case of Northern
hybridization, total RNA from the parasite was electrophoresed in
formaldehyde containing 0.8% agarose gel. 0.69-9.44-kb RNA markers
(Life Technologies, Inc.) were run and stained separately with ethidium
bromide before transfer of the gel onto the membrane. Gels were
transferred to Hybond N+ membrane (Amersham International) by capillary
blotting following membrane manufacturer's protocol. Air-dried blots
were then UV cross-linked in a UV cross-linker (Bio-Rad) for 2 min and
pre-hybridized at 42 °C for 2-3 h. Hybridization was carried out at
42 °C for 18-20 h. Blots were washed twice at 65 °C for 15 min
each with 2 × saline/sodium/phosphate/EDTA, 0.1% (w/v) SDS
followed by washes with 1 × saline/sodium/phosphate/EDTA, 0.1%
SDS and 0.1 × saline/sodium/phosphate/EDTA, 0.1% SDS, and
exposed to Fuji x-ray film for autoradiography. The quantitation was
performed using the gel-documentation system (Ultraviolet Products
Inc.) as per manufacturer's protocol by estimating the volume of the
signal.
A GST reporter-based vector (pGEX-1) was used
as an expression vector (23). A HindIII-EcoRI
restricted 138-bp amino-terminal fragment of PfP0 (46-184 bp of
GenBankTM accession number U56663[GenBank]) (7) was flushed at both ends with Klenow and subcloned in pGEX-1 restricted with
SmaI. The Escherichia coli cells harboring the
vector pGEX-1 and the recombinant containing PFP0-N insert were induced
with 1 mM isopropyl--D-thiogalactopyranoside for 2 h at 37 °C (23). The total cell lysates were run on a 12% denaturing SDS-polyacrylamide gel electrophoresis and stained with
Coomassie Blue to show the fusion protein (PfP0-N). The polyacrylamide gel piece containing the fusion protein band was cut out and crushed in
liquid nitrogen and dissolved in phosphate-buffered saline (8 g/liter
NaCl, 0.2 g/liter KCl, 1.44 g/liter Na2HPO4,
0.24 g/liter KH2PO4, pH adjusted to 7.4 with
NaOH). The resulting slurry containing an estimated amount of 100 µg
of protein was injected into two rabbits. Five boosts were given to
each animal to generate antibodies with reasonable titer (>1000 as
checked by enzyme-linked immunosorbent assay).
Pf4 cDNA
clones in
gt11, as well as wild type
gt11, were grown on E. coli strain Y1090 to lytic phase (6). Hybond-C membrane (Amersham
International) soaked in
isopropyl-
-D-thiogalactopyranoside was overlaid on these
phage plates and grown for an additional 12-16 h. The membranes were
washed three times with 1 × TBS-T (200 mM Tris, 50 mM NaCl, 0.05% Tween 20, pH 7.5). A pool of six reactive
human immune sera (1:100 dilution) used for the differential immunoscreening (6) was first incubated overnight at 4 °C with membrane saturated with the wild type
gt11 lysate. Then the sera was
incubated overnight with several membranes containing the lysate of
recombinant
Pf4. Monospecific antibodies against
Pf4 were eluted
with 5 mM glycine from the membrane, neutralized with 1 M Tris, and then dialyzed overnight with three changes of
TBS-T. Resulting monospecific antibody solution against
Pf4 was
concentrated using an Amicon concentrator (Amicon Inc.), reconstituted
to the original volume of the human sera with 1 × TBS-T, and used
for immunoprecipitation.
Asynchronous
cultures of P. falciparum containing 10-12% parasitemia
were washed twice with methionine- and cysteine-free RPMI 1640 medium
and resuspended at a final hematocrit of 5% in the same medium
supplemented with 10% human serum. 100 µCi/ml of both radiolabeled
methionine and cysteine were added to the culture medium and incubated
for 4 h at 37 °C under normal culture conditions with
occasional shaking. After labeling, the cells were washed and extracted
as described elsewhere (24). In the case of 32P-labeling, 1 mCi/ml radiolabeled orthophosphoric acid was neutralized using 1 N NaOH added to the washed parasites at 5% hematocrit (about 10% parasitemia) in the RPMI 1640 medium supplemented with 0.5% Albumax (Life Technologies, Inc.), incubated for 4 h at
37 °C, and the rest of the steps were the same as above.
Monospecific human immune sera against Pf4 as well as rabbit
polyclonal antibody against PfP0-N were used to precipitate the
35S- and 32P-labeled parasite proteins (24).
The immunoprecipitated samples were run on 12% denaturing gels, which
were stained with Coomassie Blue for 1 h and destained with
destaining solution (30% methanol, 10% acetic acid) for 2 h.
35S-Containing gels were soaked with 1M sodium
salicylate for 30 min and washed with water for another 30 min. In the
case of 32P-containing gels, the destaining was continued
overnight with several changes of the destaining solution. The gels
were then dried using Gel dryer (Hoefer Scientific Co.) and exposed to
Fuji x-ray film for autoradiography.
IFA studies were done with P. falciparum asexual (FCR3 strain) as well as gametocytic stages (NF54 strain) with polyclonal antibodies against PfP0-N as described earlier (25). Briefly, asynchronous asexual and sexual stage cultures of P. falciparum were coated on glass slides as smears and air dried for overnight. The slides were fixed with methanol for 30 s at room temperature, blocked with 100 µg/ml bovine serum albumin solution for 2-3 h at room temperature, and washed at room temperature with 1 × TBS-T for 10 min each for three times. Slides were incubated with respective antibodies for 3-4 h at room temperature. FITC-conjugated anti-rabbit IgGs (Cappel, Organon Teknika, Durham, NC) at 1:80 dilution were used as the secondary antibody. The slides were observed under a Zeiss (Axioplan) microscope using × 100 Neofluor phase contrast objective and Zeiss filter FT500/600. All antibodies were cleared with GST and E. coli crude proteins and used at 1:100 dilution.
In Vitro Inhibition of P. falciparum GrowthIgG from rabbit sera were purified by ammonium sulfate precipitation followed by batch purification with DEAE-cellulose at pH 6.5 (26). P. falciparum growth inhibition assays were performed in triplicates in 24-well sterilized tissue culture plates (Nunc, Roskilde, Denmark) in a total culture volume of 1 ml. The effect of IgG on parasite growth was assessed by starting with ring stage-synchronized parasites at a 0.2-0.5% initial parasitemia and monitoring growth over 48 h without any medium replacement. Blood mononuclear cells from healthy donors were separated on Ficoll-Hypaque density gradient (Pharmacia Biotech Inc.). The adherent monocytes were selected in 24-well plates and counted (2). In the wells containing the adherent monocytes, P. falciparum cultures were added at a ratio of approximately 200 red blood cells/monocyte. Control wells consisted of (a) culture alone, (b) culture and monocytes alone, (c) culture and control IgG, and (d) culture, control IgG, and monocytes. Test and control IgG were added at a concentration of 1.0-1.5 mg/ml of final culture volume. Parasitemia was monitored every 6 h by preparing thin smear slides from each well and by microscopic examination of >10,000 red blood cells. In each experiment, the number of rings, trophozoites, and schizonts were counted separately. Total parasitemia was estimated as the sum total of the rings, trophozoites, and schizonts.
A Southern blot of genomic DNA from the HB3 strain of P. falciparum, probed with the 251-bp Pf4 insert, showed that it
hybridizes with the 6.1-, 13-, 1.5-, and 11-kilobase pair band when the
DNA was restricted with EcoRI, ClaI,
DraI, and HaeIII, respectively (Fig.
1, panel A). The gene has an internal
EcoRI site, and this was demonstrated by probing the same
blot with a 700-bp fragment, L-4-7, representing the carboxyl-terminal
part of the protein (7), which lit up the same bands for all
restriction digests except that of EcoRI, where it showed a
7.0-kilobase pair band (Fig. 1, panel B). Southern analysis
of genomic DNA from two other P. falciparum strains, FCR3
and NF54, was also performed with these two probes, and no significant
restriction fragment length polymorphism was observed. These results
show that the PfP0 is coded by a single gene and is well conserved in
different strains of the parasite.
A Northern blot of stage-specific total RNA from the asexual stages of
P. falciparum, probed with the 251-bp Pf4 fragment, showed a dominant 3.0-kb-size fragment in every substage (Fig. 2). However, a second band, about 2.0 kb in size, was
also seen in all stages. A quantitative determination of the ratio of
these transcripts showed that the 3.0-kb message was 2.1 ± 0.26-fold as abundant as the 2.0-kb message in each of the stages.
However, the 3.0-kb transcript was about 2.0- and 1.5-fold greater in
abundance in the trophozoites compared with the rings and the schizont
stages, respectively. The coding region of the P0 gene is 957 bp.
However, the gene seems to possess a long 5
-untranslated region of
about 1.4 kb in length, as observed by the size of cDNA clones
isolated (7). Polymerase chain reaction studies using primer sequences within the coding region amplify the same size of fragments when genomic DNA is used as a template (data not shown), and therefore, there are no introns within the coding sequence. However, the presence
of introns in the 5
-untranslated region is yet to be determined.
Fig. 3A shows the expression of the
GST-fusion protein of the amino-terminal domain of the P. falciparum P0 protein (PfP0-N) in the total E. coli
cell lysate. The 46-amino acid stretch (17-62 amino acids) from the
138-bp HindIII-EcoRI fragment (7) produced the
expected 31-kDa GST-fusion protein. Polyclonal rabbit antibodies, with
a titer of >1000, were raised against PfP0-N and used for immunoprecipitation, immunofluorescence, and growth inhibition studies.
This antibody immunoprecipitated a single 38-kDa phosphoprotein from
the 35S- and 32P-labeled P. falciparum proteins (Fig. 3B). Control antibodies such
as rabbit preimmune sera and rabbit polyclonal antibodies raised
against GST did not show any band (data not shown). To ascertain that
the human immune sera originally used for the differential screen
actually recognized the PfP0 protein domain in the Pf4 expression
clone, monospecific human immune sera was affinity-purified using
Pf4 expression clone and used for immunoprecipitation analysis. This
also brought down the 38-kDa P0 protein from 35S-labeled
parasite proteins (Fig. 3B). Human immune sera
affinity-purified against the control phage
gt11 did not show any
reactivity with the parasite extract (Fig. 3B). Western
blots of the recombinant PfP0-N protein with immune and patient sera
samples showed that the recombinant protein was detected only by immune
sera samples and not by patient sera samples (data not shown).
Rabbit antibodies raised against PfP0-N were used for IFA studies with
different asexual stages (Fig. 4, A and
B) as well as gametocytic stages of the parasite (Fig. 4,
C and D). Rabbit preimmune sera and polyclonal
antibodies against GST were used as control. The control antibodies
showed no staining with parasites, whereas the anti-PfP0 antibody
showed staining with all the different developmental stages of the
parasite. In some of the gametocytic stages, intense staining of some
sub-cellular domains were noticed (Fig. 4D), but the
significance of this observation is not clear. The localization of the
antigen was predominantly intracellular, however there appeared to be
some staining at the surface as well. Preliminary experiments with
biochemically fractionated parasite extracts do show antibody
reactivity to the particulate fractions on Western blots (data not
shown). When tested on Toxoplasma gondii, an apicomplexan
parasite closely related to Plasmodium, it was observed to
stain the surface, as confirmed by double staining with surface and
internal markers of
Toxoplasma.2
It has been reported that the protective human immune sera inhibits the
in vitro growth of P. falciparum only in the
presence of monocytes (2). However, these experiments were performed with mixed specificities of immunoglobulins. To ascertain whether anti-PfP0 antibody inhibits the growth of P. falciparum in
the presence or absence of monocytes, in vitro inhibition
studies using synchronized parasites starting from the ring stages were performed (Fig. 5). The antibodies raised against PfP0-N
completely inhibited the growth of the parasites over a 48-h period
(Fig. 5, panel A). Preimmune sera and sera raised against an
irrelevant P. falciparum GST-fusion protein showed no
inhibition in the growth of the parasites. Cultures without any IgG and
with sera raised against GST protein showed normal growth (data not
shown). 4-6 independent experiments were performed for each treatment,
and the figure represents an average of these experiments. The presence of monocytes were found to stimulate the parasite growth, but the
inhibition of growth of P. falciparum by the antibody was independent of the presence of monocytes (Fig. 5A).
In vitro growth inhibition assay.
Panel A shows the effect of purified IgG from rabbit
polyclonal antibodies against PfP0-N either in the presence () or
absence (
) of human monocytes on the growth of 48-h in
vitro culture of P. falciparum FCK2 strain. Preimmune
sera with (
) or without (
) human monocytes and an antibody
against irrelevant GST-fusion protein (
) were used as controls.
Panel B shows the comparison of the percentage of rings (
), trophozoites (
), and schizonts (
) present at different time points during the same
in vitro growth inhibition assay described in panel
A, and in the presence of monocytes, The top panel
shows the distribution of substages in the presence of control
preimmune sera, and the lower panel shows the substages in
the presence of rabbit anti-PfP0-N antibody.
The growth inhibition studies were assayed by microscopically counting at least 10,000 red blood cells for each time point. The counting assay was performed rather than the hypoxanthine-uptake assay, because the uptake of hypoxanthine by the monocytes would cause errors in the assay. Also, starting with a synchronized culture and counting the different substages at every time point would show the development of the substages of the parasite with time. Cultures with synchronized ring stages were the starting point, and every 6 h the different substages of the parasite were counted. In the presence of the control antibody, the parasites progressed through the ring, trophozoite, and schizont stages in about 24 h, and subsequently there was an increase in the ring stages, indicating the invasion of fresh red blood cells (Fig. 5B, top panel). However, in the presence of anti-PfP0 antibody, even though the parasites developed from rings to trophozoite and schizont stages, fresh infection of red blood cells did not take place as the number of ring stages remained close to zero until 48 h in culture (Fig. 5B, lower panel). The same profile was observed for the distribution of the parasite substages irrespective of the presence (Fig. 5B) or absence (data not shown) of monocytes.
Earlier we had reported the cloning of the full-length gene of
PfP0 from the 7G8 strain of P. falciparum (7). The largest cDNA clone had a coding region 957 bases long, an unusually long 5-untranslated region of at least ~1.4 kb, and a 3
-untranslated region of ~50 bases, making it a total of at least ~2.4 kb in size.
The 3.0-kb transcript therefore matches with this size. It is not clear
whether the second transcript of ~2.0-kb size is a processed
transcript or a degradation product. The transcripts are twice as
abundant in the trophozoite stages, indicating that the expression of
this gene may be regulated.
The predicted molecular mass of the full-length gene sequence is 37.5 kDa. The rabbit sera raised against the recombinant protein domain of the P0 gene recognized a 38-kDa P. falciparum protein from both 35S- and 32P-labeled cultures, which matches with the expected size of the deduced P0 protein. The monospecific immune sera also recognized the same size protein. These immunoprecipitation studies showed that the antibodies raised against the PfP0-N recognize a protein of the predicted size, that it is indeed a phosphoprotein, and that antibodies against PfP0-N are present among malaria-immune people and not in patients.
The P0 protein was first identified as a member of the P family of proteins through coprecipitation studies using antibodies against the conserved carboxyl-terminal region (9, 15). Although definitive studies have been performed to demonstrate that P1 and P2 are phosphoproteins (27), there has been no direct evidence that P0 is a phosphoprotein. 32P-Labeled cultures of yeast cells showed a large number of phosphoriboproteins (28), of which the 41-kDa protein has been assumed to be the P0 protein (9). Thus, for the first time, through immunoprecipitation studies using anti-P0 antibodies, we are demonstrating that P0 is indeed a phosphoprotein.
Autoantibodies against the conserved carboxyl-terminal domain of the P proteins have been found in 10-15% of patients with an autoimmune disorder, systemic lupus erythematosus (15). These autoantibodies have been implicated as a cause for the psychotic disorders among these patients. Immune response has also been documented against P0 protein in chronic Chagas' disease (16) and leishmaniasis (17). However, in each of these cases the reactivity has been reported against the carboxyl-terminal domain. The systemic lupus erythematosus patient sera have been tested for reactivity against the amino-terminal domain of the P2 protein but did not show any response (15). The reactivity of the malaria-immune sera to P0 is widespread (87% of immune sera samples) (6) and is directed toward the Pf4 region which is the amino-terminal domain of the PfP0 protein (7). Thus this immune response toward P0 in malaria-immune persons appears to be different from that observed for the systemic lupus erythematosus patients.
The mechanism of the passive clearance of the parasites in malaria patients with IgG of malaria-immune persons is not clearly understood. The IgG may be specifically interacting and blocking crucial parasite domains, or it may be attaching to the parasitized cells and allowing other cellular components of the immune system to clear the parasites. The latter has been supported by studies in which a pool of immune human sera inhibited the in vitro growth of P. falciparum only in the presence of monocytes (2). However, these experiments were performed with mixed specificities of immunoglobulins. Anti-PfP0-N antibody inhibits the growth of P. falciparum irrespective of the presence or absence of monocytes, indicating a direct block of parasite invasion. The mechanism behind the inhibition of the P. falciparum growth with anti-PfP0-N antibodies is unclear given the predominant cytoplasmic localization of the PfP0 protein. The inhibition is unlikely to be due to the inhibition of the P. falciparum ribosomal activity as the development from the ring to the schizont stages were nearly normal in the presence of anti-PfP0-N antibody over a period of 24 h. The growth inhibition by anti-PfP0-N IgG clearly seems to act at the erythrocyte-invasion step. While the IFA results do indicate a surface component in addition to the cytoplasmic localization of P0 in the trophozoites and gametocytic stages, whether there is a surface localization of this protein in the merozoite stage is yet to be determined. Interestingly, the presence of an antigenic determinant related to the carboxyl-terminal of P0 protein has been localized to the surface of hepatoma, neuroblastoma, and human fibroblast cells (29). What this surface determinant of P0 protein may be and what role it may play in the erythrocyte invasion by the malarial parasite remains to be established.
We are indebted to Nirbhay Kumar and Cheryl Ann Lobo for help with the supply of gametocytes and general immunoprecipitation techniques. We thank Namita Surolia for the FCK2 strain of P. falciparum and Ruchira Jaitly, a visiting summer student, for help with the expression of PfP0-N fusion protein.