From the Medical Entomology Section and the
¶ Malaria Genetics Section, Laboratory of Parasitic Diseases,
NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425
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ABSTRACT |
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In Plasmodium-infected mosquitoes,
oocysts are preferentially located at the posterior half of the
posterior midgut. Because mosquitoes rest vertically after feeding, the
effect of gravity on the ingested blood has been proposed as the cause
of such a biased distribution. In this paper, we examined the oocyst
distribution on the midguts of mosquitoes that were continuously
rotated to nullify the effect of gravity and found that the typical
pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the
posterior part of the posterior midgut. We examined whether the
distribution of oocysts depends on the distribution of vesicular ATPase
(V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised
against recombinant Aedes aegypti V-ATPase B subunit
indicated that the majority of V-ATPase-overexpressing cells in
Ae. aegypti and Anopheles gambiae are localized
at the posterior part of the posterior midgut. We propose that the
typical distribution of oocysts on the mosquito midgut is
attributable to the presence and the spatial distribution
of the V-ATPase-overexpressing cells in the midgut epithelium.
Designing rational strategies to block transmission of malaria can
benefit from understanding how the Plasmodium parasite has
adapted to the mosquito midgut cellular environment (1). Until now, the
cellular and biochemical basis of Plasmodium development in
the mosquito has been poorly understood. During most of their development in a vector mosquito, malaria parasites remain associated with the midgut. The posterior midgut is the primary site of storage of
ingested blood, of the absorption of salts and fluids, and of the
digestion of blood components. Within a day after ingestion, the
parasites develop in the posterior midgut as motile ookinetes that
escape from the blood bolus and invade the epithelium. Invasion of the
mosquito midgut is obligatory for further development and transmission.
After escaping from the invaded cells, the ookinetes remain associated
with the midgut under the basal lamina for about 1 week and are then
transformed into oocysts, which leads to the development of sporozoites
that are infectious to the vertebrate host.
In Plasmodium-infected mosquitoes, the oocysts are primarily
distributed at the posterior portion of the posterior midgut. The
anterior portion has relatively fewer oocysts, especially when the
infection rate is low (<100 oocysts/gut). Previously, this biased
distribution was explained by the fact that mosquitoes rest on a wall
for a day or two after feeding, their abdomen being in a vertical
position. It has therefore been proposed that, because of gravity, the
ingested blood cells and parasites settle at the bottom portion of the
midgut and that ookinetes only get a chance to invade this part of the
midgut (2-4).
One argument against the above explanation comes from the finding that
diuresis rapidly compacts the blood meal long before the ookinetes
invade the midgut epithelium (5), thereby reducing the possibility for
the ookinetes to settle down. Here, we examine whether gravity is
indeed involved in determining the distribution of oocysts on the
mosquito midgut.
The mosquito midgut is a complex tissue composed of a variety of cell
types (6). We have recently found that a specific type of cell (Ross
cells) is preferentially invaded by Plasmodium gallinaceum
ookinetes (7). Ross cells are less basophilic than other cells in the
epithelium, they are devoid of microvilli on their surface, and their
cytoplasm appears less osmiophilic by electron microscopy. They also
overexpress a vesicular ATPase (V-ATPase).1 Because of the
parasite's preferential invasion of Ross cells, the development of the
parasite was thought to possibly depend on the presence, number, and
location of these cells in the midgut.
We have cloned the Aedes aegypti V-ATPase B subunit cDNA
and used the recombinant protein to prepare an antibody to investigate the distribution of V-ATPase-overexpressing cells in the midgut of
Ae. aegypti and Anopheles gambiae. The oocyst
distribution was studied in mosquitoes kept in a device designed to
nullify the effect of gravity, and the location of P. gallinaceum ookinete invasion of the midgut epithelium was
assessed at the peak invasion period. We provide evidence that Ross
cells, rather than gravity, determine the typical distribution pattern
of the oocysts in infected mosquitoes.
Mosquitoes and Parasites--
Ae. aegypti black-eye
strain and An. gambiae G3 were raised under standard
conditions (26 °C, 80% humidity, photoperiodism (12 h dark-12 h
light)) and fed on diluted Karo syrup (CPC International Inc.,
Englewood Cliffs, NJ). P. gallinaceum (8A strain) was
maintained by serial subpassages in 4-5-week-old white Leghorn chickens.
Detection of the Distribution of Oocysts on Mosquito
Midguts--
Adult mosquitoes were fed on an infected chicken for 20 min. Fed mosquitoes were transferred into two cylindrical containers (9 cm high, 9 cm in diameter). One container was kept on an immobile rack,
thus allowing mosquitoes to rest on the wall. The other container was
rotated for 48 h at a speed of 10 rpm. The rotation prohibited any
specific orientation of the abdomens of the blood-fed mosquitoes. The
mosquitoes were then kept in an insectary and dissected for oocyst
counting at 8 days post-blood meal. Oocysts were counted separately in
the anterior and posterior halves of the posterior midgut with a
microscope equipped with an eyepiece micrometer. The statistical
comparison (nonparametric) and the significance test of the oocyst
numbers between two groups of infected mosquitoes were determined using
Wilcoxon rank sum analysis. All of the mosquitoes in the test and
control groups that received a blood meal were included in analysis
regardless of whether they had oocysts.
Preparation of the Mosquito V-ATPase-specific Probe--
Two
degenerate primers designed from the predicted amino acid sequence of
the V-ATPase B subunit from yeast (8) were as follows: sense primer,
5'-GG(ACGT)TT(TC)CC(ACGT)GGITA(TC)ATGTA(TC)A-3' (degeneracy = 1/128); and antisense primer,
5'-GC(TC)TC(TC)TCICCIAC(ACGT)AC(ACGT)GC(TC)TTC-3' (degeneracy = 1/128). Thirty-five cycles of PCR were performed with these primers
using 1 ng of Ae. aegypti genomic DNA as target. The
conditions for the PCR were 30 s at 94 °C, 30 s at
45 °C, and 1 min at 72 °C, performed in an automatic thermal
cycler (GeneAmp PCR System 9600, Perkin-Elmer). The amplified product
was subcloned into the pCR2.1 cloning vector (Invitrogen, Carlsbad, CA)
and its nucleotide sequence determined.
A cDNA library made with the mRNA of unfed and blood-fed
midguts of Ae. aegypti and the Lambda Zap Express cloning
vector (Stratagene, La Jolla, CA) was a kind gift of Dr. Anthony James (University of California at Irvine). The unamplified library consisted
of 12.9 × 106
For cloning, the ORF of the Ae. aegypti V-ATPase B subunit
cDNA was amplified by PCR with a 5' primer containing the start codon ATG and an antisense 3' primer containing the stop codon TAA.
NdeI and BamHI sites were added for directional
cloning. The conditions for the PCR were the same as above, except that the extension time was 3 min. The 1.5-kilobase ORF was cloned into the
pET11a bacterial expression vector (Stratagene) and introduced into
E. coli BL21(DE3) cells (Stratagene). Expression of the
recombinant protein was induced by adding 1 mM
isopropyl-
To produce antiserum, the Coomassie Blue-stained recombinant V-ATPase B
subunit band from a preparatory SDS-polyacrylamide gel was excised,
homogenized with Freund's complete adjuvant in PBS, and used to
immunize a New Zealand White rabbit (Spring Valley Laboratories,
Sykesville, MD). Subsequent boosts, performed at 14-day intervals, were
done with incomplete adjuvant. After the third boost, the serum was
tested by Western blot analysis. To deplete E. coli-specific
antibodies, the antiserum was preabsorbed twice with heat-denatured and
sonicated extracts of E. coli BL21(DE3).
Western Blot Analysis with Mosquito Proteins--
Midguts from
sugar-fed, blood-fed, and infected blood-fed mosquitoes were dissected
in PBS and heated at 80 °C for 30 min in Laemmli sample buffer.
Chicken blood (negative control) was treated the same way. After
SDS-polyacrylamide gel electrophoresis, proteins were transferred to
nitrocellulose (Nytran, Schleicher & Schuell). Membranes were blocked
overnight with 5% milk in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) and probed with a 1:100 dilution of the
preabsorbed polyclonal antiserum. Blots were developed using an
alkaline phosphatase-conjugated goat anti-rabbit antibody (dilution
1:500, Kirkegaard & Perry Laboratories, Gaithersburg, MD) and the
Western Blue kit (Promega, Madison, WI).
Immunofluorescence Assay--
To detect V-ATPase-overexpressing
cells, sugar-fed mosquitoes were dissected 4-6 days after emergence.
Gut tissues were immediately fixed in 4% paraformaldehyde, 2 mM MgSO4, 1 mM EGTA, and 0.1 M PIPES buffer (7). Fixed tissues were dehydrated by
incubating 5 min each with 20, 40, 60, and 80% methanol in PBS, pH
7.2, and then for 15 min with 100% methanol. Tissues were rehydrated
by sequentially washing them with decreasing concentrations of methanol in PBS. Rehydrated and permeabilized tissues were blocked overnight at
4 °C with PBTG (1% bovine serum albumin, 0.1% Triton X-100, and
2% goat serum in PBS) and incubated overnight at 4 °C with preabsorbed V-ATPase antiserum diluted in PBTG. After three washes (1 h
each) with PBTG, the tissues were incubated for 4 h with a
fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Pierce) diluted in PBTG and 0.005% bisbenzimide H33258 fluorochrome (nuclear stain, Calbiochem).
To examine the early invasion of the midgut epithelium by ookinetes,
P. gallinaceum-infected blood-fed mosquitoes were treated the same way, except that the blood-fed midguts were dissected in PBS
42 h post-feeding and cut longitudinally to remove the blood meal.
An additional incubation with a rhodamine-labeled anti-Pgs28 monoclonal
antibody (dilution 1:500) was performed to detect the parasites that
had invaded the epithelium. The tissues were then washed five times
with PBTG (1 h each), mounted, and examined with a Leitz Ortholux 2 fluorescent microscope using appropriate filter blocks for rhodamine
and fluorescein isothiocyanate. In a defined area of the mosquito
tissues, the total number of cells, the number of
V-ATPase-overexpressing cells, and the number of ookinetes that invaded
the epithelium were determined, respectively, by nuclear staining,
V-ATPase staining, and Pgs28 staining.
Distribution of P. gallinaceum Oocysts on the Mosquito
Midgut--
In a preliminary experiment, 12 infected blood-fed
mosquitoes were individually kept head down in small tubes. After
48 h, which is enough for the food bolus to concentrate in the
midgut epithelium and for the majority of ookinetes to invade the
midgut epithelium, the mosquitoes were transferred into a cage until dissected and examined for the distribution of oocysts. The
distribution of oocysts in these mosquitoes remained biased toward the
posterior portion of the posterior midgut (data not shown), similar to
the oocyst distribution in the control mosquitoes. We repeated the experiment with 60 fully engorged mosquitoes that were rotated for
48 h to avoid orientation in a particular direction. A control group of mosquitoes from the same cohort, fed on the same infected chicken, was kept in a cage and allowed to rest vertically after feeding. Eight days later, oocyst distributions were determined for the
midguts of mosquitoes from both groups. No significant difference
(p > 0.1) was observed in the mean number of oocysts per midgut between the control (74.2 ± 15.8, n = 13) and test (102.9 ± 8.6, n = 60) groups. This
result suggests that the rotation does not significantly influence the
total number of oocysts per midgut; however, the difference between the
mean numbers of oocysts present on the anterior part (32.6 ± 2.8)
and on the posterior part (70.3 ± 6.3) of the posterior midgut
was highly significant (p < 0.001) in the rotated
mosquitoes. This finding indicates that gravity does not determine the
oocyst distribution. Indeed, all of the mosquitoes in the control group
had a majority of the oocysts distributed toward the posterior part of
the posterior midgut as expected (Fig. 1;
see also Fig. 5A). Of the 60 mosquitoes that were rotated,
only two had slightly more oocysts on the anterior part of the
posterior midgut. In four other mosquitoes, the oocysts were equally
distributed between the anterior and the posterior parts of the
posterior midgut. All of the other mosquitoes (90%) had oocysts
primarily in the posterior half of the posterior midgut. The lack of
difference in the distribution of oocysts between the control and test
mosquitoes suggests that gravity does not have a detectable effect on
oocyst distribution in the posterior midgut of the mosquito.
In Vivo Invasion of P. gallinaceum Ookinetes in Ae. aegypti
Midguts--
To determine the stage at which the spatial distribution
of oocysts is defined, we examined the early invasion of ookinetes in
the mosquito midgut. The number of parasites that invaded the midgut
epithelium was estimated at 42 h after infection, when the
majority of invasion is complete but no oocysts are yet formed (9). The
invaded parasites were detected on the surface of intact midguts using
rhodamine-labeled monoclonal antibodies raised against the ookinete
surface protein Pgs28 (10). The majority of parasites were found in the
posterior half of the posterior midgut, whereas only a few invasion
events were detected in the anterior half (see Fig. 5, B and
C), suggesting that the oocyst distribution is determined at
or prior to the time of invasion. Because in Ae. aegypti the
parasites preferentially invade a particular type of cell known as Ross
cells (7), we examined whether the distribution of these cells in the
mosquito midgut could determine the location of the oocysts. One of the
characteristics of Ross cells is their overexpression of a V-ATPase. We
therefore chose to use the Ae. aegypti V-ATPase as a marker
to examine the distribution of these specialized cells in the mosquito midgut.
Spatial Distribution of V-ATPase-overexpressing Cells in the
Posterior Midgut of Mosquitoes--
The sequence of the B subunit of
V-ATPase enzymes is well conserved among organisms as evolutionarily
separated as bacteria and humans. We prepared two degenerate primers
from the amino acid sequence of the yeast V-ATPase B subunit, ranging
from amino acid 303 to 310 for the 5' primer and amino acids 417 to 424 for the 3' primer (8). A PCR product of the expected size (365 base
pairs) was amplified from genomic DNA prepared from newly emerged
larvae of Ae. aegypti. Data base comparisons using the sequenced PCR product demonstrated 100% identity at the amino acid
level with V-ATPase B subunit sequences from the dipterans Culex
quinquefasciatus (11) and Drosophila melanogaster (12) and the lepidopterans Manduca sexta (13) and
Helicoverpa virescens (14). This identity suggests that the
amplified DNA fragment derives from the Ae. aegypti V-ATPase
B subunit gene.
A specific digoxygenin-labeled probe made from this PCR-amplified gene
fragment was used to screen an Ae. aegypti midgut cDNA library prepared from a pool of sugar-fed and uninfected blood-fed adult mosquito midgut mRNA. Nine positive clones were identified and found by sequence analysis to contain the PCR probe sequence. The
complete sequence of one of these cloned cDNAs was determined by
primer walking. This cDNA is 2538 base pairs long and contains a
1491-base pair ORF encoding a 496-amino acid protein (Fig.
2). The calculated mass of the protein is
55,221 Da and is within the predicted size of V-ATPase B subunits from
other organisms (range 54-58 Da). A Prosite analysis identified the
signature sequence for the V-ATPase B subunit (PPVNVLPSLS; amino acids
376-385). The predicted amino acid sequence is 98.4% identical with
the C. quinquefasciatus V-ATPase B subunit (11), 97.8%
identical with the D. melanogaster protein (12), and 97.0%
identical with the M. sexta protein (14). This sequence also
shares high similarities (50-95%) with its counterparts from
organisms as different as bacteria, fungi, plants, parasites, and
vertebrates (Fig. 3).
The ORF from the cloned Ae. aegypti V-ATPase B subunit
cDNA was amplified by PCR and inserted downstream of the T7
promoter of the pET11a vector between the NdeI and
BamHI sites. This recombinant plasmid (pSC1) was then
introduced into E. coli BL21(DE3). After induction with
isopropyl-
A polyclonal rabbit antiserum was raised to Ae. aegypti
V-ATPase B subunit recombinant protein. The antiserum was preabsorbed with heat-denatured and sonicated extracts of the parent E. coli BL21(DE3) strain. As determined by Western blot, this
preabsorbed serum recognized the recombinant protein (Fig. 4,
lane 3) but no other E. coli or chicken blood
antigens (Fig. 4, lanes 3 and 4). Therefore, the
preabsorbed antiserum was used for all subsequent experiments.
To determine whether the native mosquito V-ATPase B subunit was
recognized by the antiserum, we performed a Western blot analysis with
mosquito proteins prepared from sugar-fed adult Ae. aegypti midguts. One polypeptide of the same estimated size as the recombinant protein was detected in the midgut tissue (Fig. 4, lane 5),
suggesting that the antiserum primarily recognizes the mosquito
V-ATPase. Protein extracts prepared from midguts of sugar-fed
mosquitoes and uninfected or infected mosquitoes 24 h after the
blood meal were compared by Western blot analysis (Fig. 4, lanes
5-7). An equivalent of two midguts was used in each case. Within
the sensitivity range of the Western blot, no difference was noted in
the intensity of the bands between these differently fed midguts. This
indicates that blood feeding or infection with Plasmodium
does not affect V-ATPase expression in the mosquito midgut.
We then used immunofluorescence assay to determine whether the
antiserum detects V-ATPase-expressing cells in sugar-fed Ae. aegypti midguts. As seen in Fig. 5,
E and G, the antiserum recognizes a subset of
cells in the mosquito midgut. This is similar to our previous finding
with a C. quinquefasciatus V-ATPase antiserum (7).
We have examined the distribution of V-ATPase-overexpressing cells in
the posterior midgut of Ae. aegypti. Most notable during this analysis was the nonrandom distribution of these cells in this
region; ~90% were present at the posterior portion of the posterior
midgut versus ~10% at the anterior part of the posterior midgut (Fig. 5, D and E).
V-ATPase-overexpressing Cells and P. gallinaceum Ookinete Invasion
in An. gambiae Midguts--
An. gambiae G3 mosquitoes are
vectors for human malaria but are considered to be refractory to
P. gallinaceum development (15). However, in a small
proportion of these mosquitoes, a few parasites develop as oocysts,
again at the posterior half of the posterior
midgut.2 In our previous
attempts with Culex V-ATPase antiserum, we failed to detect
V-ATPase-overexpressing cells in An. gambiae midguts, precluding the study of whether cells similar to Ross cells were present in Anopheles mosquitoes. Here, we examined whether
Ae. aegypti V-ATPase antiserum can detect
V-ATPase-overexpressing cells in An. gambiae. By Western
blot, we detected a single protein of similar size to the one found in
Ae. aegypti (Fig.
6A). We then used the
antiserum in an immunofluorescence assay to detect cells that express
the enzyme. Similar to Ae. aegypti, the antiserum specifically recognized V-ATPase-overexpressing cells in the An. gambiae midgut epithelium (Fig. 6D). Because our
V-ATPase antiserum was able to detect the Anopheles enzyme
and the cells that express this enzyme, we examined whether P. gallinaceum also invades V-ATPase-overexpressing cells in
An. gambiae and whether the oocyst distribution is
determined by the presence of these cells.
An. gambiae were fed with P. gallinaceum-infected
blood, and 36-40 h later, their midguts were dissected and probed with
V-ATPase B subunit and Pgs28 antisera. We found that, as in Ae.
aegypti, P. gallinaceum ookinetes invade An.
gambiae cells that express high levels of V-ATPase (Fig.
6B). When the distribution of V-ATPase-overexpressing cells
was examined, we found that the anterior half of the posterior midgut
had very few of these cells (less than 10% of the total V-ATPase-overexpressing cells) and that more than 90% of them were
present at the posterior half. These data indicate that although An. gambiae G3 is refractory to P. gallinaceum,
P. gallinaceum ookinetes do invade functionally related
cells in both Ae. aegypti and An. gambiae.
In this paper, we provide evidence that the distribution of
Plasmodium oocysts on the posterior midgut of mosquitoes is
related to the presence and distribution of V-ATPase-overexpressing
Ross cells. We also present evidence indicating that gravity plays no
major role in the phenomenon. Indeed, when mosquitoes were kept under
conditions in which gravity could not have any effect on the
distribution of the ingested blood cells and parasites in the posterior
midgut, we found that the pattern of oocyst distribution remains biased
toward the posterior end of the posterior midgut. In only 10% of
mosquitoes did we find oocysts distributed in equal or greater numbers
on the anterior half of the posterior midgut (Fig. 1). The reasons for
this unusual distribution pattern are unclear but may involve
individual differences in the vector or alternative distribution of
Ross cells or other invaded cell types in the midgut. We have also
shown that the initial invasion of the midgut epithelium by ookinetes
primarily occurs at the posterior part of the posterior midgut. When
the distribution of V-ATPase-overexpressing cells in Ae.
aegypti and An. gambiae was examined, we found that these cells were also primarily located at the posterior part of the
posterior midgut.
V-ATPases are members of the class of ATP-hydrolyzing proton pumps
ubiquitous among living organisms (16). The ATPase family can be
divided into three distinct groups (17): i) the P-ATPases, which are
localized in plasma membranes; these are composed of a single subunit,
and the degradation of ATP involves a phosphorylated intermediate; ii)
the F0/F1-ATPases, which are multisubunit
molecules found in eubacteria and in organellar membranes (mitochondria and chloroplasts); and iii) the V-ATPases, which were first identified in vesicular membranes but have recently been found in the cytoplasmic membrane of epithelial cells. They resemble the
F0/F1-ATPases both in their structure and in
the way they catalyze ATP but are classified as a distinct family
because they are not sensitive to F0/F1-ATPase
inhibitors. V-ATPases are composed of nine subunits organized in two
domains: one transmembrane domain and one cytoplasmic (catalytic)
domain. The catalytic domain includes three copies of both the A
(catalytic) and B (regulatory) subunits. Taking advantage of the high
degree of sequence conservation among organisms as different as
bacteria, fungi, plants, insects, and vertebrates, we have cloned and
expressed the B subunit of the Ae. aegypti V-ATPase. The
production of an antiserum raised against the recombinant protein
allowed us to study both Ae. aegypti and An.
gambiae cells that overexpress this enzyme.
The role of V-ATPases in the mosquito midgut is not known. In M. sexta larvae midguts, specialized cells resembling vertebrate goblet cells overexpress V-ATPase and appear to be involved in ion and
water transportation from the midgut lumen to the hemolymph (18). It
has also been shown that malpighian tubules, which play an important
role in fluid absorption, overexpress a V-ATPase at their surface (19).
It might therefore be possible that the V-ATPase-overexpressing cells
present in the posterior midgut of mosquitoes are involved in the
diuresis that occurs immediately after blood feeding. However, it is
not known why the parasites prefer to use these cells to cross the midgut.
This study was undertaken in the context of how the human malaria
parasite Plasmodium falciparum develops in its vector and is
transmitted. P. gallinaceum is better adapted to use in the laboratory and hence is commonly used as a model for malaria
transmission-blocking studies. Several genes have been cloned from
P. gallinaceum and reveal greater homology with P. falciparum than with any other Plasmodium spp. (20).
Also, the blocking of P. gallinaceum oocyst development in
Aedes mosquitoes by the chitinase inhibitor allosamidin can
be reproduced with P. falciparum in Anopheles
mosquitoes (21). Therefore, the results presented here might apply to
P. falciparum, allowing us to initiate work on
V-ATPase-overexpressing cells in An. gambiae to determine
their role in human malaria transmission.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phages, of which 95.3% were
recombinant as determined by blue/white screening. This library was
amplified before screening (final titer = 2.3 × 109 plaque-forming units/ml in Escherichia coli
XL1-Blue cells). The library was screened with a digoxygenin-labeled
PCR-amplified fragment of the Ae. aegypti V-ATPase B subunit
gene (see above). The preparation, hybridization, and detection of the
probe were performed using the DIG/Genius kit (Boehringer Mannheim).
Nine positive clones were purified and excised in vivo to
produce pBK-CMV recombinant phagemids (Stratagene). Once the identity
of the clones was confirmed by initial sequencing, the complete
nucleotide sequence of one clone was performed by primer walking using
a modified dideoxynucleotide-based sequencing method (ABI model 377;
Applied Biosystems, Foster City, CA).
-D-thiogalactopyranoside into the growth
medium and was assessed by SDS-polyacrylamide gel electrophoresis on
cells harvested 3 h post-induction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Distribution of P. gallinaceum
oocysts on midguts of undisturbed or rotated Ae.
aegypti. Each line links the number of oocysts on the
anterior part (Ant.) of the posterior midgut to the number
of oocysts on the posterior part (Post.) of the posterior
midgut of a single mosquito. Rotated mosquitoes were kept for 48 h
post-blood meal in a rotating device.
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Fig. 2.
Nucleotide and deduced amino acid sequences
from Ae. aegypti V-ATPase B subunit cDNA. The
1491-base pair ORF starts with the initiation codon at position 235 and ends with a stop codon at position 1723. The signature sequence for
V-ATPase A and B subunits is underlined (amino acids
376-385).
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Fig. 3.
Sequence comparison of the Ae.
aegypti V-ATPase B subunit with V-ATPase B subunits from
evolutionary diverse organisms: insects (D. melanogaster), vertebrates (Homo sapiens),
plants (Arabidopsis thaliana), nematodes
(Caenorhabditis elegans), fungi (Neurospora
crassa), and parasites (P. falciparum). Alignments were performed using the
Clustal method. Dots represent amino acids that are
identical to the mosquito sequence. Dashes represent gaps
introduced to maximize the alignments.
-D-thiogalactopyranoside, the transformed E. coli produced high levels of the recombinant protein
(Fig. 4, lane 2). No
expression was detected in uninduced transformed bacteria (Fig. 4,
lane 1) or in induced nontransformed bacteria (data not
shown). The estimated mass of the recombinant protein is similar to the
predicted size of the protein encoded by the ORF of the cloned
cDNA.
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Fig. 4.
Expression of recombinant Ae.
aegypti V-ATPase B subunit cDNA in a bacterial
expression vector and of the native protein in Ae.
aegypti. The PCR-amplified, full-length cDNA was
cloned into the pET11a expression vector and expressed into E. coli BL21(DE3) as described under "Experimental Procedures."
Lane 1, uninduced transformed bacteria; lane 2,
induced transformed bacteria. A strong band, with relative migration at
55 kDa, is present and highly expressed only in the induced bacteria
(arrow). The band was used to prepare an antiserum that was
preabsorbed with the parent bacterial strain and used in Western blot.
Lane 3, recombinant V-ATPase (Rec. V-ATPase;
positive control); lane 4, chicken blood (negative control);
lane 5, sugar-fed (Sug) Ae. aegypti
midguts; lane 6, blood-fed (Bld) Ae.
aegypti midguts; lane 7, infected (Inf)
blood-fed Ae. aegypti midguts. No notable difference in the
intensity of expression was detected in any of these last three
samples. IPTG,
isopropyl- -D-thiogalactopyranoside.
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Fig. 5.
P. gallinaceum ookinete invasion and oocyst
development in the Ae. aegypti posterior midgut.
A, oocyst distribution 8 days post-blood meal. The
arrowhead points to the middle of the Ae. aegypti
posterior midgut. The closed arrow identifies the junction
of the anterior and posterior midgut, and the open arrow
shows the junction of the posterior midgut and the hindgut.
B, ookinete invasion 42 h post-blood meal in the
anterior half, and C, in the posterior half of the posterior
midgut. V-ATPase-overexpressing cells at D, the anterior
half, and E, the posterior half of the posterior midgut.
F, the same field as in G but with staining for
epithelial cell nuclei. G, magnification of the boxed
area in E.
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Fig. 6.
Detection of V-ATPase and of cells that
overexpress this enzyme in An. gambiae posterior
midguts. Sugar-fed (Sug), blood-fed (Bld),
and infected blood-fed (Inf) An. gambiae midguts
were treated as described under "Experimental Procedures."
A, the Ae. aegypti antiserum recognizes a sharp
band of 55 kDa. B, immunofluorescence assay studies reveal a
colocalization of V-ATPase-overexpressing cells with the invasion of a
P. gallinaceum ookinete. The biased distribution of the
V-ATPase-overexpressing cells toward the posterior end of the posterior
midgut can be seen in C-F: C, V-ATPase staining
of the anterior part of the posterior midgut; D, V-ATPase
staining of the posterior part of the posterior midgut; E,
total cells in the same area as C; F, total cells
in the same area as D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Anthony A. James for permission to use the Ae. aegypti midgut cDNA library. We are indebted to Drs. Louis H. Miller and José M. C. Ribeiro for their support and critical comments throughout the work. We thank Dr. David C. Kaslow for the Pgs28 antiserum. We acknowledge the technical assistance of André Laughinghouse and Kevin Lee in rearing mosquitoes and cultivating parasites. We also thank Brenda Rae Marshall for preparing the manuscript.
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FOOTNOTES |
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* This work was supported, in part, by Grant 950476 (to Louis H. Miller) from the United Nations Developmental Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF092934.
§ Supported by the Vector Biology Network of the John D. and Catherine T. MacArthur Foundation.
To whom correspondence should be addressed: Laboratory of
Parasitic Diseases, NIAID, National Institutes of Health, Bldg. 4, Rm.
B2-39, 9000 Rockville Pike, Bethesda, MD 20892-0425. Tel.: 301-496-9389; Fax: 301-402-8536; E-mail:
mohammed_shahabuddin{at}nih.gov.
2 S. O. Cociancich and M. Shahabuddin, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: V-ATPase, vesicular ATPase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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