Diagnosis of toxoplasmosis using a synthetic glycosylphosphatidyl-inositol glycan

Article abstract of DOI:10.1002/anie.201406706

Around 2 billion people worldwide are infected with the apicomplexan parasite Toxoplasma gondii which induces a variety of medical conditions. For example, primary infection during pregnancy can result in fetal death or mental retardation of the child. Diagnosis of acute infections in pregnant women is challenging but crucially important as the drugs used to treat T. gondii infections are potentially harmful to the unborn child. Better, faster, more reliable, and cheaper means of diagnosis by using defined antigens for accurate serological tests are highly desirable. Synthetic pathogen-specific glycosylphosphatidyl-inositol (GPI) glycan antigens are diagnostic markers and have been used to distinguish between toxoplasmosis disease states using human sera.

Full text of DOI:10.1002/anie.201406706

Angewandte
Chemie
DOI: 10.1002/anie.201406706
Diagnostic GPI
Diagnosis of Toxoplasmosis Using a Synthetic Glycosylphosphatidyl-
inositol Glycan**
Sebastian Gçtze, Nahid Azzouz, Yu-Hsuan Tsai, Uwe Groß, Anika Reinhardt,
Chakkumkal Anish, Peter H. Seeberger, and Daniel Varꢀn Silva*
Abstract: Around 2 billion people worldwide are infected with
the apicomplexan parasite Toxoplasma gondii which induces
a variety of medical conditions. For example, primary infection
during pregnancy can result in fetal death or mental retardation
of the child. Diagnosis of acute infections in pregnant women is
challenging but crucially important as the drugs used to treat
T. gondii infections are potentially harmful to the unborn child.
Better, faster, more reliable, and cheaper means of diagnosis by
using defined antigens for accurate serological tests are highly
desirable. Synthetic pathogen-specific glycosylphosphatidyl-
inositol (GPI) glycan antigens are diagnostic markers and have
been used to distinguish between toxoplasmosis disease states
using human sera.
unborn child, leading to mental and physical retardation and
fetal death.[6] Congenital toxoplasmosis can be prevented by
stopping parasite transmission from mother to fetus using
potentially harmful chemotherapy. Thus, false-positive diag-
noses of the disease are to be avoided. Currently, diagnosis of
acute toxoplasmosis relies on serological detection of IgG and
IgM antibodies as well as determination of IgG avidity against
T. gondii antigens. The IgM titer has the greatest diagnostic
value, as the absence of IgM antibodies can rule out a recently
acquired infection with greater certainty. However, commer-
cially available test kits used by nonreference laboratories are
known to generate high rates (up to 60%) of false-positive
test results.[7] Therefore, the identification of antigens that are
suitable for the development of reliable diagnostic tools is
important.
Glycosylphosphatidylinositols (GPIs) are a class of complex
molecules that are present in high abundance (about 106 copies
per cell) on the cell surface of T. gondii.[8] These glycolipids are
found in all eukaryotic organisms and share the conserved
glycan core structure a-Man-(1!2)-a-Man-(1!6)-a-Man-(1!
4)-a-GlcNH2-(1!6)-myo-Ino (Figure 1).[9] T. gondii uses GPIs
to anchor enzymes and other surface proteins via their C-
terminus to the extracellular side of the plasma membrane. Two
main GPI glycoforms that are T. gondii specific contain an
additional branch connected to the O4-position of ManI.
Whereas GPI 1 bears an a-Glc-(1!4)-b-GalNAc side chain,
GPI 2 lacks the glucose moiety.[10] Molecules 1 and 2 also differ
in their function. While GPI 2 serves as a membrane anchor for
surface antigens and enzymes, GPI 1 occurs free on the cell
surface without being covalently attached to any protein.[11] In
particular, GPI 1 is an immunologically active compound that
induces an early IgM response in humans after infection with
T. gondii.[12] We hypothesized that the phosphoglycans of GPIs
1 and 2, as well as substructures and biochemical intermediates,
display potential diagnostic markers that may enable the
differentiation between acute and latent toxoplasmosis.[13]
The isolation of homogeneous GPIs from parasites is
challenging and does not give access to GPI derivatives or
substructures. We employed chemical synthesis to obtain
GPIs[14] of defined structure and high purity that are suitable
for biochemical assays and medical applications.[15] This
approach also excludes the possibility of false-positive test
results due to cross-reactivity against impurities in T. gondii
isolates.[16] In order to elaborate diagnostic tools based on
carbohydrate microarrays[17] of GPI-glycans, an orthogonal
handle that ensures regioselective covalent attachment to
a solid support was required. To preserve the free amine
groups of GPIs, a thiol was selected as a nucleophilic
orthogonal functionality. Therefore, a linear alkane thiol
T
he apicomplexan T. gondii is a parasite that is capable of
infecting all warm-blooded animals.[1] Infection occurs
through the ingestion of contaminated raw meat or oocysts
shed by cats, the parasiteꢀs primary host.[2] Although T. gondii
infections are the third leading cause of food-borne infections
requiring hospitalization in the USA,[3] it is not perceived as
a major public health problem in developed countries, owing
to its usually benign etiopathology.[4] However, in immuno-
compromised individuals, latent toxoplasmosis can lead to
dangerous medical conditions such as toxoplasmic encepha-
litis, a severe inflammation of the brain and a leading cause of
death in AIDS patients.[5] Primary infection during pregnancy
can cause transmission of the parasite from the mother to the
[*] Dr. S. Gçtze, Dr. N. Azzouz, Dr. Y.-H. Tsai, A. Reinhardt, Dr. C. Anish,
Prof. Dr. P. H. Seeberger, Dr. D. Varꢀn Silva
Department of Biomolecular Systems
Max Planck Institute of Colloids and Interfaces
Am Mꢁhlenberg 1, 14424 Potsdam (Germany)
and
Institute of Chemistry and Biochemistry
Freie Universitꢂt Berlin
Arnimallee 22, 14195 Berlin (Germany)
E-mail: daniel.varon@mpikg.mpg.de
Prof. Dr. U. Groß
Nationales Konsiliarlabor Toxoplasma und Institut fꢁr Medizinische
Mikrobiologie, Universitꢂtsmedizin Gçttingen (Germany)
[**] We thank the Max Planck Society and the RIKEN–Max Planck Joint
Research Center for Systems Chemical Biology for generous
financial support. A.R. thanks the “Studienstiftung des Deutschen
Volkes” for a PhD scholarship. Y.-H. T. thanks the DAAD for a PhD
scholarship. We thank Dr. Ryan McBride for the protocol employed
in the detection of anti-carbohydrate antibodies in serum samples.
We thank Felix Brçcker and Andreas Geißner for critically reading
this manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201406706.
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phitylation of alcohol 9[19] by an in situ
generated mixed anhydride of H-phospho-
nate 10[18] and pivaloyl chloride. Subsequent
oxidation of the resulting H-phosphonate
using iodine in wet pyridine furnished phos-
phate salt 11 in 87% yield. Acidic hydrolysis
of the silyl ether gave access to primary
alcohol 12. A one-pot procedure yielded
bisphosphate 13 through phosphitylation of
12 using H-phosphonate 14[14d] followed by
oxidation and final hydrazinolysis of the
levulinic ester in 76% yield. For global
deprotection of the remaining benzyl ether
units 13 was submitted to Birch reduction
conditions to furnish carbohydrate antigen 3
in 55% yield.[15g]
Figure 1. Conserved pseudopentasaccharide core structure of GPIs and T. gondii specific
glycolipids 1 and 2. GlcNH2 =d-glucosamine, Ino=inositol, Man=d-mannose.
phosphodiester was installed at the O1-position
of myo-Ino instead of the naturally occurring
lipid moiety to provide conjugation-ready phos-
phoglycans 3–6 (Figure 2).[18]
To investigate the immune response against
the carbohydrate portion of 1 and 2 upon
infection with T. gondii and determine the
immunodominant epitopes, such as side-
branch glycosylation or phosphorylation
status, the glycans 3 and 4, as well as a set of
closely related sugars, were synthesized. Glycan
5 represents a biosynthetic precursor of GPI 2
Scheme 1. Synthesis of phosphoglycan 3. Reagents and conditions: a) 1. 10, PivCl, Py;
2. I2, H2O, 87%; b) Sc(OTf)3, MeCN, H2O, 508C, 76%; c) 1. 14, PivCl, Py; 2. I2, H2O;
3. hydrazine, 76%; d) NH3(l), THF, Na(s), ꢀ788C, tBuOH, 55%. Bn=benzyl,
Cbz=carboxybenzyl, Lev=levulinyl, Piv=pivaloyl, Py=pyridine, Tf=triflyl, TIPS=tri-
isopropylsilyl.
that is not phosphorylated at the O6-position of
ManIII.[10] Trisphosphorylated compound
6
reflects a minimal structure of mammalian
GPIs, which possess an additional phosphoe-
thanolamine and a GalNAc branch at the O2-
and O4- positions of ManI respectively.[15b]
To complete the collection of synthetic GPI glycans
Access to the complex GPI molecules was based on the
convergent route for the synthesis of these glycolipids and
their derivatives we had developed earlier.[19] To illustrate the
syntheses of glycans 3–6[20] the preparation of 3 is shown in
detail (Scheme 1). The synthesis commenced with the phos-
related to toxoplasmosis antigens, the substructures 15–17
(Figure 3A) displaying the side branch of GPI 3 were also
prepared according to the general synthetic strategy.[20] Linear
glycans 18 and 19,[18] which mirror GPI anchors found on the
apicomplexan parasites Plasmodium falciparum and Crypto-
sporidium parvum,[21] as well as
pseudodisaccharide
20[18]
(Fig-
ure 3B) were synthesized and
placed on the GPI-glycan arrays to
examine
a
possible
immune
response against the conserved
backbone. Mannan-related tetra-
mannoside 21[22] (Figure 3C) served
as a control carbohydrate as it is not
related to the GPIs of T. gondii but is
found in certain types of mycobac-
teria[23] and fungi.[24]
All carbohydrates bearing either
a single amine or thiol linker were
printed on epoxide-modified glass
slides and covalently immobilized on
the surface.[25] The resulting carbo-
Figure 2. GPI-glycans 3–5 resemble the carbohydrate moieties of T. gondii GPIs. Compound 6
represents the minimal structure of all mammalian GPIs.
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side 21 in detectable concentrations;
these are not attributed to an infection
with T. gondii, but possibly other patho-
gens.[28]
In contrast, all sera from patients
diagnosed with acute toxoplasmosis
showed high levels of IgG and IgM
antibodies recognizing the full glycan
structure 3 (Figure 4B). In addition
IgG and IgM antibodies directed
against substructures 15–17 of GPI
1 were also found in almost all samples
from acutely infected persons exhibit-
ing signal intensities comparable to
those obtained for phosphoglycan 3.
Most acute-toxoplasmosis samples con-
tained IgG and IgM antibodies that
bind phosphoglycan 4, but the intensity
of the signals was usually lower com-
pared to those generated by the a-Glc
containing compounds 3, 15, 16, and 17.
IgG and IgM antibodies binding to
linear GPI 19 and monophosphorylated
GPI-glycan 5 were detected in some
sera of acutely infected patients. Anti-
bodies recognizing the P. falciparum
GPI-glycan structure 18 were not
observed in any acutely infected
Figure 3. Carbohydrate structures evaluated as markers for the diagnosis of toxoplasmosis.
A) Substructures 15–17 represent the side branch of GPI 3. B) Linear phosphoglycans 18–20
contain conserved features of GPIs. C) Mannan substructure 21 is present in the cell wall of
pathogenic yeast.
hydrate microarrays were incubated with reference sera of
patients suffering from acute or latent toxoplasmosis as well
as of seronegative individuals utilizing a highly reproducible
protocol for the detection of anti-carbohydrate antibodies in
humans.[26]
The screening results (Figure 4) showed that all sera from
noninfected patients[27] contained undetectable or low levels
of IgG and IgM antibodies directed against the printed GPIs
or their substructures (Figure 4A). Some healthy individuals
exhibited IgG and IgM antibodies that bound to tetramanno-
patients. Interestingly, individual sera also contained IgM
but no IgG antibodies directed against pseudodisaccharide
20.
As anticipated, samples from latently infected patients
showed an IgG antibody binding pattern and signal intensities
that are comparable to the analyses of sera from acutely
infected humans (Figure 4C). In contrast, IgM levels against
all printed structures were considerably reduced.
These results are in accordance with reports describing
that the immune response against GPIs in T. gondii infected
humans is mainly directed against the a-Glc-
containing glycolipid 1 and not GPI anchor 2,
which indicates that the side-branch motif in
this molecule is an important antigenic epi-
tope.[11] None of the tested samples contained
antibodies that bind to mammalian glycan 6,
suggesting that the adaptive immune system
is able to distinguish self from non-self GPI
structures through the additional phospho-
ethanolamine unit at ManI.
As antibodies against GPI-glycan 3 were
detected in all infected samples, this structure
was evaluated as a diagnostic marker for
toxoplasmosis (Figure 5). A statistical analy-
sis showed that mean IgG as well as IgM
serum antibody levels against saccharide 3
are significantly increased during the acute
phase of the infection. While the average
concentration of IgG in the blood does not
considerably decrease after the acute phase,
IgM levels drop to values that are in most
Figure 4. Representative scans of a carbohydrate microarray incubated with generic
serum samples (dilution 1:15) from patients having A) no infection, B) acute toxoplasmo-
sis, and C) latent toxoplasmosis (printing pattern is superimposed). D) Scheme describ-
ing the positions of the compounds on the microarray.
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Antigen 3 emerged as a suitable biomarker for the
diagnosis of different stages of toxoplasmosis. The IgG level
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differentiate latent and acute toxoplasmosis.[29]
In conclusion we synthesized GPI-glycans found on the
surface of parasite and mammalian cells. Parasite-specific
carbohydrates presented on glycan arrays enabled a detailed
investigation of the immune response against GPIs upon
natural infection with T. gondii in humans. Screening of
human sera revealed that only the full phosphoglycan of GPI
1 displays a valuable antigen recognized by all infected serum
samples tested. Analysis of GPI 3 specific IgG and IgM levels
allowed for distinction between infected and noninfected
individuals. Thus, parallel detection of IgG and IgM directed
against the single synthetic cell-surface antigen, GPI 3,
constitutes a suitable strategy for the development of
a faster and more-specific diagnostic tool to discriminate
between latent and acute toxoplasmosis.
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Received: June 30, 2014
Published online: && &&, &&&&
Keywords: biomarkers · carbohydrate microarrays ·
.
medicinal chemistry · toxoplasmosis
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Communications
Diagnostic GPI
S. Gçtze, N. Azzouz, Y.-H. Tsai, U. Groß,
A. Reinhardt, C. Anish, P. H. Seeberger,
D. Varꢁn Silva*
&&&& — &&&&
Diagnosis of Toxoplasmosis Using
a Synthetic Glycosylphosphatidylinositol
Glycan
Good news for cat lovers! A glycosyl-
phosphatidylinositol (GPI) glycan resem-
bling an antigen on the parasite T. gondii
was synthesized and immobilized on
a microarray. This setup can be used to
diagnose toxoplasmosis. Latent and
acute toxoplasmosis can be readily dis-
tinguished.
6
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