A small, heat stable chromophore extracted from
mosquitoes has recently been implicated as the signal that induces
mating of Plasmodium, the malaria parasite. We have used
high resolution electrospray mass spectrometry to determine that this
gamete activation factor (GAF) has a m/z = 205.0450, suggesting a molecular species composition of
C10H7NO4. Xanthurenic acid (XA), a
product of tryptophan catabolism, was determined to have an elemental
composition, ultraviolet absorbance maxima, and mass spectrum
consistent with those characteristics of GAF. XA activated
gametogenesis of Plasmodium gallinaceum and P. falciparum in vitro at concentrations lower than 0.5 µM in saline buffered to pH 7.4. A structural analog of
XA, kynurenic acid (C10H6NO3), also
activated gametogenesis but only at higher concentrations and with less
effect. We propose that XA is GAF. This is the first evidence that XA
has induction activity.
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INTRODUCTION |
Species of Plasmodium, the parasitic protozoa that
cause malaria, must undergo one sexual recombination in each
transmission cycle. Although the mature sexual stages, or gametocytes,
are present in the blood of infected vertebrate hosts, mating only occurs in the gut lumen of vector mosquitoes. This process of gametogenesis begins immediately after infected blood reaches the gut
and is completed within about 20 min. During that brief time the
parasite dissolves two erythrocyte membranes surrounding it and, in the
case of the male gamete, completes a profound nuclear reorganization
that results in the formation of several flagellar, free swimming
gametes (1). It has been postulated that the signal that activates
these dramatic events is in the mosquito gut (2). We recently confirmed
the existence of such a signal and demonstrated that it is a heat
stable chromophore of Mr
205 (3). We report
here the results of further chemical analyses that demonstrate that
gamete activation factor
(GAF)1 is
xanthurenic acid (XA), a tryptophan metabolite. Although XA is widely
present in both vertebrates and invertebrates, this is the first
evidence that it has inductive activity. It is likely this finding will
play a part in the future development of drugs aimed at impeding the
transmission of malaria, the most widespread and destructive parasitic
disease in the world.
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EXPERIMENTAL PROCEDURES |
Preparation of Extracts--
Procedures for extracting and
chromatographically purifying GAF from Anopheles stephensi
mosquito tissues have been described (3). In this case GAF was prepared
from 75,000 mixed male and female heads of A. stephensi
(batch NP30); storage of the final extract was in water at
20 °C.
Analytical Procedures--
High resolution electrospray
interface mass spectrometry of GAF was performed at the Mass
Spectrometry Laboratory of the National Center for Environmental Health
at the Centers for Disease Control and Prevention (Atlanta, GA).
Positive ion spectra were obtained from a Micromass 70-4F double
focusing magnetic sector tandem mass spectrometer fitted with an
electrospray interface. Solvent delivery was performed with HP1090 HPLC
equipped with a HP diode array detector. A Vydac C18 column
(1 × 150 mm, 90 Å, 5 µm) was used at a flow rate of 40 µl
min
1. HPLC solvents used were water/0.1% trifluoroacetic
acid (Buffer A) and acetonitrile/0.1% trifluoroacetic acid (Buffer B).
Gradient elution was obtained by a hold at 1% Buffer B for 1 min and
then a linear ramp from 1 to 13% Buffer B in 19 min followed by a
linear ramp to 30% Buffer B in 5 min (for eluting leucine enkephalin time marker). The effluent was monitored at 205 nm. Wavelength scans
(200-600 nm) were taken for all peaks eluting from the column. Initially, low resolution scans (1000 resolution at 10% valley) were
performed over the m/z range of 100-600 atomic mass units. The high resolution scans were performed over a narrow mass range to
bracket the major ion (m/z = 206) with two mass
reference ions from PEG-200. The mass calibrant was injected
post-column just prior to and just after elution of the 206 ion to
negate the effect of ion drift on mass measurement. The analyses were
done in triplicate. Potential elemental compositions were deduced
employing the standard Micromass OPUS software, and candidate compounds
were suggested from a search of National Institute of Standards and
Technology software (1994-1995 edition) supplied with the OPUS
system.
UV spectra and retention time of XA were determined under identical
conditions of reverse phase high performance chromatography as
mosquito-derived GAF. The system employed consisted of a Vydac narrow
bore column (2.1 mm × 25 mm, 5 µm, 300 Å) attached to Beckman 126 pumps and a model 168 Diode Array Detector controlled by Beckman System Gold software (version 8.1). The compounds were eluted isocratically with a solvent of 5% CH3CN/94.9%
H2O/0.1% trifluoroacetic acid at a flow rate of 0.3 ml min
1 and ambient temperature.
XA (Sigma) concentrations were determined from the molar extinction
coefficient (
) at 247 nm. The
247 was found by
dissolving 0.54 µmol (0.11 mg) of XA in 1 ml (0.54 mM) of
PBS (pH 7.4) and measuring the absorbance of the solution in a Beckman
DU-65 spectrophotometer. The
247 = 20,988 cm
1 M
1. Concentrated solutions
of XA were prepared in water and then adjusted to pH 7.4 with 0.5 n NaOH before addition of PBS. GAF concentrations were estimated
from a standard curve of XA peak areas measured by RP-HPLC. Kynurenic
acid (Sigma) stock solution was prepared by dissolving 0.12 mg (0.58 µmol) of the monohydrate form in 1 ml (0.58 mM) of PBS.
Kynurenine (Sigma, 0.5 H2O mol/mol) 0.32 mg (1.5 µmol)
was dissolved in 1 ml of PBS (1.5 mM). Kainic acid
monohydrate (Sigma) 0.47 mg (2 µmol) was dissolved in 1 ml of PBS (2 mM).
Bioassays--
In vitro activity was determined using
P. gallinaceum, a bird malaria parasite, and cultured
P. falciparum, a human parasite, as described previously
(3). Briefly, 5 µl of test substances dissolved in PBS were added to
preparations of gametocytes stored in PBS, which retards gametogenesis;
induction is scored by microscopically counting the number of male
gametes exflagellating 15-30 min later.
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RESULTS |
We had previously determined that GAF has a major, singly charged
ion species at m/z 206. Three runs of high resolution
voltage scans were done of the bioactive fraction over a narrow range bracketing 206 (Table I). Results were
highly consistent, and a median weight of 206.0450 was chosen to do an
elemental composition search, with limits on the accuracy of the mass
measurement set at 20 ppm. Only compounds containing carbon, hydrogen,
oxygen, nitrogen, phosphorus, or sulfur were searched; several elements with distinctive isotope distributions not evident in spectra (e.g.. chlorine, bromine, and copper) were excluded from
consideration. The most likely composition, at an accuracy of 0.6 ppm,
was C-10, H-8, N-1, O-4; assuming that the observed ion was
[M+H]+, the most likely composition of the parent species
was C-10, H-7, N-1, O-4. A search of the National Institute of
Standards and Technology electron ionization libraries done for all
entries with nominal weight of 205 yielded 292 candidate compounds;
only those with the formula C-10, H-7, N-1, O-4 were chosen for further consideration. All but four candidates could be eliminated on the basis
of having weights outside the 20 ppm limit, having rare atoms unlikely
to be present (e.g. silicon or boron), or having atoms known
to be absent (e.g. chlorine or bromine). The four compounds
included 2-isobenzazol and three isomers of quinolinecarboxylic acid,
one of which, XA, commonly occurs in insects (4).
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Table I
High resolution mass measurements on GAF
GAF purified from A. stephensi heads was analyzed by
accurate mass high resolution electrospray interface mass spectrometry.
Three runs were performed employing narrow range voltage scans using
PEG-200 for mass calibration (details are under "Experimental
Procedures"). Measured weights are averages of two readings.
Resolutions are for 10% valley.
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We found the chromatographic (Fig.
1A) and UV (Fig.
1B) characteristics of XA, or of a mixture of XA and GAF, to
be identical to those of GAF. The ability of XA to stimulate
gametogenesis in P. gallinaceum was comparable with that of
GAF, with a threshold dose as low as 0.5 µM (Table
II). XA and GAF at 1 mM also
activated the human parasite P. falciparum at the same rates
(73 and 51, respectively); XA concentrations as low as 325 nM induced prolific gametogenesis. A close analog of XA,
kyneurenic acid, activated gametogenesis of P. gallinaceum
but at a maximum effect about one-third that of XA and with no
induction below 100 µM (Table II). Two other products of
tryptophan catabolism, kainic acid and kynurennine, had no
bioactivity.

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Fig. 1.
Analytical comparison of GAF and XA.
A, chromatographic profiles of GAF (0.112 nmol) isolated
from mosquito heads; XA (0.116 nmol); and a mixture of GAF (0.112 nmol)
and XA (0.116 nmol). The method for RP-HPLC through a narrow bore
C18 column is detailed under "Experimental Procedures."
B, normalized UV absorbance profiles of GAF (solid
line) from mosquito heads and XA (dashed line) from
RP-HPLC runs. Note that the nearly complete superimposition of the two
sets of data give the appearance of a single line. Inset,
chemical structure of XA.
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Table II
The ability of XA and KYNA to induce gametogenesis of the malaria
parasite P. gallinaceum compared with that of the natural signal, GAF
Induction was measured by counting at 200× magnification the number of
male parasites exflagellating in a 5-µl suspension of washed,
infected chicken erythrocytes 15-30 min after addition of test
compounds in PBS. Data are means of triplicate matched tests; the
negative control, PBS, was always 0. Statistical significance was
calculated between compounds at the same concentrations; each marked
value is significantly different from value immediately below. XA
activated the human parasite P. falciparum at 325 nM (data not shown). ND, not done.
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DISCUSSION |
Gametocytes rarely constitute more than 5% of all infected red
blood cells, and unlike the asexual forms, whose rapid cell division
and growth force disintegration of the host erythrocyte independent of
environmental cues, the gametocytes depend on an external signal to
activate escape. The survival benefit to the parasite of this
requirement is clear: only mating in the gut of the vector will ensure
transmission to a new host. A reduction in temperature from 37 °C to
less than 30 °C is a precondition for activation but does not alone
induce gametogenesis (1). We have recently shown that a single
molecular species, GAF, present in both the gut and the head of a
vector mosquito acts as a potent inducer of gametogenesis in a simple,
protein-free, saline solution that otherwise would indefinitely
prohibit activation (3). In this report we present compelling
analytical evidence that XA is GAF. The bioactivity of XA is
confirmatory.
XA is widely distributed in insects, where it is a byproduct of the
2,3-dioxygenase pathway of tryptophan degradation, which leads through
kynurenine to, among other compounds, the ommochromes, common pigments
of invertebrate eyes and integument (4). In vertebrates, XA has been
routinely measured in urine as an indication of vitamin B6
levels (5) and has been linked indirectly to some pathological
conditions, such as cataract formation (6). It has not, however, been
reported before to have inductive ability. How it induces gametogenesis
is not yet known. Before gametogenesis begins, the parasite is
separated from the erythrocyte cytoplasm by the host cell-derived
parasitopherous vacuole membrane and from the external environment by
the erythrocyte plasma membrane (7). There is evidence that
gametogenesis is preceded by activation of the phosphoinositide cascade
(8) and release of intracellular bound Ca2+ (9). XA, which
is relatively hydrophilic, may act as a ligand at the outer,
erythrocyte membrane. Notably, KYNA, which differs structurally from XA
only in the absence of a hydroxyl group from position 8, also
stimulated gametogenesis, although at a substantially lower level. KYNA
is now known to be an endogenous, nonspecific antagonist at the
excitatory amino acid receptors in vertebrate central nervous systems,
as well as having a high affinity for the glycine site in the
N-methyl-D-aspartate receptor complex (10); XA
has been used as an inactive control in studies of KYNA kinetics.
Presumably the mosquito head has proved to be a relatively rich source
of GAF because of the metabolism of eye pigment, but it is not yet
clear why XA is also present in its gut. Nor is the possible
contribution of XA in vertebrate serum known; few data on serum levels
have been published. Nevertheless, we now have a tool for investigating
the precise sequence of gametogenesis induction. Considerable effort is
now focused on developing vaccines targeted at the sexual stages in the
gut, which would block malaria transmission (11), and it seems likely
that understanding the mechanics of induction will make possible a
parallel effort to develop transmission blocking drugs.
We appreciate the technical support provided
by A. Laughinghouse and K. Lee (National Institutes of Health), V. Maggio (Centers for Disease Control), J. Glass and M. Dowler (Walter
Reed Army Institute of Research), and C. Paul (Bethesda Research
Institute). L. Tsai (National Institutes of Health) provided valuable
advice.