FEMS Microbiology Letters 247 (2005) 8190

www.fems-microbiology.org

The putative chitin deacetylase of Encephalitozoon cuniculi: A

surface protein implicated in microsporidian spore-wall formation
Damien Brosson a, Lauriane Kuhn b, Gerard Prensier a, Christian P. Vivare`s a,
Catherine Texier a,*
a

Equipe Parasitologie Moleculaire et Cellulaire, LBP, UMR CNRS 6023, Universite Blaise Pascal, 63177 Aubie`re CEDEX, France
b
Laboratoire de Chimie des Proteines, DBMS, CEA, 38054 Grenoble, France
Received 2 March 2005; received in revised form 20 April 2005; accepted 21 April 2005
First published online 3 May 2005
Edited by R.C. Staples

Abstract
Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They dierentiate into
resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of
the brillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on
the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised
against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the
ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the
polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA
is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identication of an EcCDA
homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme
among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore
wall formation and spore resistance.

2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Microsporidia; Encephalitozoon cuniculi; Chitin deacetylase

1. Introduction
The obligate intracellular Microsporidia are sporeforming amitochondriate unicellular eukaryotes that
can parasitize a wide variety of animals ranging from
protists to mammals. Some species, including Encephalitozoon cuniculi, are opportunist human pathogens [1]
with a high seroprevalence in immunocompetent popu*

Corresponding author. Tel.: +33 4 73 40 74 34; fax: +33 4 73 40 76

70.
E-mail address: [email protected] (C. Texier).

lations [24]. Phylogenetic analyses based on some conserved proteins suggest that Microsporidia are related to
fungi [5,6]. Complete sequencing of the E. cuniculi genome [7] has also provided data consistent with a fungal
origin of Microsporidia [8].
Microsporidian spores display an original invasion
mechanism, involving a coiled structure (the polar tube)
that can be quickly extruded to transfer the spore content or sporoplasm into a target cell. The parasite enters
a proliferation phase or merogony and the resulting
meronts then undergo a dierentiation step (sporogony).
They transform into sporonts (beginning of cell wall

0378-1097/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.04.031

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D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

formation), then into sporoblasts (beginning of polar

tube elaboration) and nally into mature spores protected by a thick wall. The microsporidian spore wall
plays a major role in the protection against environmental stresses, allowing long-term survival of the parasite
after its release from the host cell [9,10]. While the classical concept of the parasite penetration mode involves
the polar tube acting as a needlesyringe system, microsporidian spores may also be internalized via attachment
to the host plasma membrane then endocytosis [11]. If
this occurs, the induced endocytosis process should depend on interactions with the wall.
The microsporidian spore wall usually comprises a
proteinaceous outer electron-dense layer (exospore)
and a chitinous inner electron-transparent layer (endospore) [1214], which is of brillar nature and connected
to the plasma membrane [13,15]. The stippled deposit of
an electron-dense material on the outer side of the parasite plasma membrane is the main visible feature of the
transition from merogony to sporogony [16,17]. This deposit turns into a continuous layer in young sporoblasts,
and should be at the origin of the exospore. The electrontransparent endospore is built in later stages and thus appears as a cytological marker of the mature spore.
Few studies have been dedicated to the characterisation of spore wall proteins. Immunostaining with polyclonal antibodies raised against partially puried E.
cuniculi proteins has revealed a 30-kDa antigen within
the exospore and a 33-kDa one close to the spore plasma
membrane [18]. Several monoclonal antibodies have
also been reported to be specic to spore wall antigens
[1921]. To date, the knowledge of the primary structure
of such antigens is limited to two exospore components
with unknown function: SWP1 (50 kDa) in both E.
cuniculi [22] and Encephalitozoon intestinalis [23], and
SWP2 (150 kDa) in E. intestinalis only [23]. Here, we
provide evidence for an E. cuniculi surface protein
belonging to the polysaccharide deacetylase family that
should be active in chitin deacetylation during all sporogonic stages.

2. Materials and methods

2.1. Microsporidian spore production and protein
extraction
The E. cuniculi spores were produced on Madin
Darby Canine Kidney (MDCK) or Human Foreskin
Fibroblast (HFF) cells as previously described [18].
Parasites (1091010) were disrupted in lysis buer containing 100 mM DTT, 4% CHAPS and 0.2% SDS, by
repeated cycle of freezingthawing and sonication (Deltasonic 1320, 300 W, 28 kHz). Disrupted spores were
then incubated in the extraction solution (7 M urea,
2 M thiourea, 100 mM DTT, 4% CHAPS, 0.2% SDS)

for 6 h at room temperature. Soluble proteins were collected and stored at
20 C.
2.2. Recombinant ECU11_0510 protein expression
The ECU11_0510 protein was expressed as a truncated
form in fusion with Glutathione-S-transferase (GST) at
the N-terminal and an 8xHis-tag at the C-terminal
extremity. PCR primers were designed (5 0 -CGGAATTCTTTGTGGACGGTCCTGTC-3 0 and 5 0 -CGCTCGAGTCCTATGCTCTCAACGCC-3 0 containing an EcoRI
and a XhoI restriction site, respectively) to amplify a 624bp genomic DNA fragment corresponding to the amino
acid region 32239 of the 254-amino acid ECU11_0510
protein. PCR reactions were performed according to
standard conditions with an annealing temperature of
55 C, and the puried product was cloned in frame with
GST into the prokaryotic expression vector pGEX-4T1
modied to provide a C-terminal 8xHis-tag after the XhoI
site. The resulting recombinant plasmid was introduced in
the Escherichia coli BL21+ strain. After induction with
IPTG, bacterial proteins were extracted with Laemmli
solution and analysed by SDSPAGE.
2.3. Gel electrophoresis and Western blot analysis
Protein samples were analysed by SDSPAGE on
12% polyacrylamide gels. For two-dimensional (2-D)
gel electrophoresis, protein isoelectrofocalisation was
performed along linear immobilized pH gradient strips
of 7 and 17 cm (BioRad) using a rehydration buer
(7 M urea, 2 M thiourea, 4% CHAPS) supplemented
with 2 mM tributyl phosphine (TBP) and 0.5% ampholytes (BioRad) with the IPGPhor apparatus (Amersham). Before standard SDSPAGE on 12%
polyacrylamide gel, strips were equilibrated with
50 mM TrisHCl pH 8.8, 6 M urea, 30% glycerol, 2%
SDS containing 100 mM DTT, and then 135 mM iodoacetamide. For mass spectrometry analysis, gels were
xed in a 7.5% acetic acid/30% ethanol solution, stained
with Coomassie Blue (BioRad) and destained with 30%
ethanol. For immunoblotting studies, proteins were
transferred onto polyvinylidene diuoride (PVDF)
membrane (Millipore). After blocking for 1 h in PBS5% skim milk, membranes were incubated for 3 h with
appropriate dilutions of polyclonal mouse antibodies
in PBS 0.1% Triton X-100, treated with a 1:10000
dilution of alkaline phosphatase-conjugated goat antimouse IgG (H + L) (Promega) for 1 h at room temperature and revealed with NBT/BCIP (Promega).
2.4. Tryptic digestion for mass spectrometry analysis
Protein spots were manually excised from Coomassie
blue-stained 2-D gels and transferred in 96-wellmicrotitration plates. Sample preparation was fully

D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

automated (Mass Prep Station Multiprobe II, Micromass). Briey, excised gels were washed with destaining
solutions (25 mM NH4HCO3 for 15 min and 50% (v/v)
acetonitrile containing 25 mM NH4HCO3 for 15 min).
After dehydration with 100% acetonitrile and drying,
proteins were reduced in 25 mM NH4HCO3, 10 mM
DTT for 1 h at 37 C, and alkylated in 25 mM
NH4HCO3, 55 mM iodoacetamide for 30 min at 37 C.
After washing with destaining solutions and pure water,
gels were shrunk again with 100% acetonitrile. Depending
on protein amount, 23 lL of 0.1 lg/lL modied trypsin
(Promega, sequencing grade) in 25 mM NH4HCO3 were
added over the gel spots for 30 min. Seven lL of 25 mM
NH4HCO3 were then added to cover the gel spots and
digestion allowed to continue for 5 h at 37 C.
2.5. MALDI-TOF-MS analysis and database search
For MALDI-TOF-MS analysis, 0.5 lL of trypsin
peptide mixture was mixed with 0.5 lL of a-cyano-4hydroxycinnamic acid at half saturation in 60% acetonitrile/0.1% (v/v) triuoroacetic acid (TFA). The resulting
solution was automatically spotted on the target plate
and rinsed by 2 lL of 0.1% TFA for 30 s, liquid being
then blown o by pressurized air. Peptide mixtures were
analysed with a MALDI-TOF mass spectrometer (Autoex, Bruker Daltonik) in reector mode over a mass
range of 04200 Da. For each sample, spectrum acquisition and annotation were done automatically. Spectra
from 200 shots at several positions were combined to generate a peptide mass ngerprint for database searches.
Spectra were calibrated using a mixture of angiotensin
II (1046.54 Da), substance P (1347.74 Da), bombesin
(1619.82 Da) and ACTH (1839) (2465.20 Da) as an
external standard. The E. cuniculi specic database was
explored with an intranet version of Mascot 1.7. All peptide masses were assumed to be monoisotopic and
[M + H]+ (protonated molecular ions). Peptide modications allowed during the search were cysteine carbamidomethylation, protein N-acetylation and methionine
oxidation. The maximum number of missed cleavages
was set to 1 and the mass tolerance to 150 ppm.
2.6. Polyclonal antibody production
Antiserum to E. coli expressed recombinant protein
was produced in BALB/c mice from crushed SDS
PAGE protein bands. Every two weeks, mice were injected intraperitoneally with samples homogenized with
Freunds adjuvant (Sigma). Serum was collected 2 weeks
after the last injection and stored at
20 C.

83

hyde in 0.1 M phosphate buer pH 7.2. After washing,

the pellet was impregnated with a cryoprotective agent
(25% glycerol, 5% DMSO in phosphate buer) and frozen in pasty nitrogen before storage in liquid nitrogen.
The 85 nm ultrathin sections were performed with the
FCS system on the Leica UltraCut S ultramicrotome.
The sections were picked on collodion-coated nickel
grids and stored on 2.3 M sucrose before immunolabelling. After blocking for 30 min with 2% BSA in PBS,
sections were incubated for 1 h30 with various dilutions
of polyclonal antibodies, and then for 1 h with a 1:100
dilution of protein A or goat anti-mouse antibody conjugated with 5 nm colloidal gold particles (Sigma). After
washing, the grids were stained and embedded with
0.5% aqueous uranyl acetate 1.6% methylcellulose for
10 min prior to examination with a JEOL 1200EX transmission electron microscope.
2.8. Sequence analysis
Physico-chemical parameters of the protein sequence
were calculated with ProtParam (www.expasy.org). The
SignalP program version 2 (www.cbs.dtu.dk/services/
SignalP/), the TMPRED program (www.ch.embnet.org/
software/TMPRED_form.html) and the PSORT II algorithm (www.psort.nibb.ac.jp/form2.html) were used to
predict signal peptide, transmembrane helices and protein localisation, respectively. Consensus secondary
structure prediction were performed on NPS@ Network
Protein Sequence Analysis (http//npsa-pbil.ibcp.fr) [24]
using the DSC, GOR4, PHD and SOPMA algorithms.
Search for protein homologies and protein domains were
done using the BLAST program (www.ncbi.nlm.nih.gov)
and the Pfam database (www.sanger.ac.uk/Software/
Pfam/search.shtml), respectively. Multiple sequence
alignments were performed with Dialign program
(bibiserv.techfak.uni-bielefeld.de/dialign/) and analyzed
for amino acid identity and similarity with Boxshade
(www.ch.embnet.org/sofware/BOX_form.html).
3. Results and discussion
Mouse polyclonal antibodies (PAb) were previously
raised against several SDSPAGE separated E. cuniculi
spore proteins and analysed by Western blotting and
indirect immunouorescence [18]. One of these, directed
against a 33-kDa protein band (anti-33 kDa PAb),
showed a labelling close to the outer face of the parasite
plasma membrane. This prompted us to identify the
protein target of this anti-33 kDa PAb.

2.7. Transmission electron microscopy immunolabelling

3.1. The anti-33 kDa PAb recognizes the ECU11_0510

protein

E. cuniculi-infected MDCK cells were pelleted, then

xed with 4% paraformaldehyde and 0.1% glutaralde-

To get a better separation of the targeted protein, we

performed 2-D electrophoresis of proteins extracted

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D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

from E. cuniculi spores (Fig. 1(a)). After Western blotting, the anti-33 kDa PAb highlighted two major regions
at 33 and 55 kDa (Fig. 1(b)). MALDI-TOF mass spec-

trometry data obtained with the 33-kDa region (Fig.

1(c)) matched the ECU11_0510 protein (SWISS-PROT:
Q8SU65) in the E. cuniculi proteome database deduced

Fig. 1. Migration on 2-D gel and identication by mass spectrometry of the anti-33 kDa PAb target. E. cuniculi spore proteins were separated by 2-D
electrophoresis and either stained by Coomassie Blue (a) or subjected to Western blot immunodetection with a 1:4000 dilution of the anti-33 kDa
PAb (b). Trypsin digests of the 33-kDa spot (c) and the 55-kDa spot (d) was analyzed by MALDI-TOF-MS. Seven (nine) experimental peptides for
the 33-kDa spot (55-kDa one), labelled with stars in (c) (in (d)), matched the ECU11_0150 protein with a score of 60 (100) and sequence coverage of
29% (33.8%) (e). Only one miscleavage was observed. Dierences between experimental and calculated molecular weights (MW) do not exceed
0.09 Da.

D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

from genome sequencing [7] (Fig. 1(e)). Identical data

were obtained with the 55-kDa spot (Fig. 1(d) and
(e)). We produced a recombinant form of the EcCDA
protein which was slightly truncated to exclude predicted transmembrane helices (see below). The puried
recombinant protein reacted with the anti-33 kDa PAb
(Fig. 2) and was also used to produce specic PAb in
BALB/c mice (anti-11_0510 PAb). These new antibodies
exhibited a similar reaction pattern as the anti-33 kDa
PAb either on the recombinant ECU11_0510 protein
or on E. cuniculi extract (Fig. 2).
3.2. The ECU11_0510 protein is predicted to be a chitin
deacetylase (EcCDA)
As supported by genome data [7], the ECU11_0510
open reading frame is a 765-bp single-copy gene located
on chromosome 11 which codes for a 254-amino acid
protein (28.1 kDa, pI 4.56). As summarized in Fig.
3(a), the extreme N-terminal region displays the characteristics of a signal peptide with a predicted cleavage site
between positions 15 (Gly) and 16 (Ser) corroborated by
the TMPred prediction of a transmembrane helix
(TMH) in the 118 region. An additional TMH should
be located at the 234253 C-terminal extremity in an
outsideinside orientation, suggesting that the protein
attached to the membrane via this TMH has a large
extracellular domain. This is in accordance with the ecient recovery of ECU11_0510 (not shown) when using
an improved membrane protein solubility mixture characterized by the amidosulfobetaine ASB14 detergent
and high chaotrope molarity [25,26]. PSORT program

Fig. 2. Immunoblotting with anti-33 kDa PAb and anti-11_0510 Pab

against both the recombinant ECU11_0510 protein and E. cuniculi
extract. The 56-kDa recombinant form of the ECU11_0510 protein
(fused in N-terminal with GST and in C-terminal with 8xHis tag) was
injected into BALB/c mice to produce polyclonal antibodies (anti11_0510 PAb). Both antibodies reacted in a similar way (CB:
Coomassie Blue).

85

also predicts an extracellular location including cell wall

(66.7%, k = 9/23).
The ECU11_0510 protein contains the PfamA conserved domain 15022 representative of the polysaccharide deacetylase family (Fig. 3(b)). This family includes
Rhizobium NodB (nodulation protein B), endoxylanases
and fungal chitooligosaccharide deacetylases. The presence of chitin in the microsporidian spore wall has been
unambiguously demonstrated [12,13]. Moreover, chitin
is the sole polysaccharide that metabolism reconstruction
predicts to be synthesized in E. cuniculi [7]. N-acetylglucosamine 6-P is converted into N-acetylglucosamine
1-P, then into UDP-N-acetylglucosamine via the activities of phosphoacetylglucosamine mutase and UDPGlcNAc pyrophosphorylase. Chitin synthase carries
out the polymerisation of N-acetylglucosamine into chitin chains. We thus assume that the ECU11_0510 protein
may be a chitin deacetylase enzyme (EcCDA) which
deacetylates chitin into chitosan. Enzymatic assays with
a puried recombinant protein that was expressed in E.
coli, were unsuccessful. The recombinant protein may
lack some post-translational modications required for
biological activity as it is the case for the yeast CDA2p
which loses its activity after deglycosylation [27].
Interestingly, strong homologues of the chitosan pathway enzymes, including CDA, were identied in the partially sequenced genome of an insect Microsporidia,
Nosema locustae renamed Antonospora locustae [28]
(Nosema locustae Genome Project, Marine Biological
Laboratory at Woods Hole, funded by NSF award number 0135272, http://jbcp.mbl.edu/Nosema/index.html).
The two microsporidian CDA sequences are of nearly
identical size and exhibit 40% identity (Fig. 3(b)).
The presence of CDA in Microsporidia provides an
additional support to the fungal origin of these intracellular parasites. Chitin deacetylases have been characterized in several fungi [29], including zygomycetes (Mucor
rouxii [30,31]) and ascomycetes (Colletotrichum lindemuthianum [32], Saccharomyces cerevisiae [33,34]). These
enzymes are monomeric glycoproteins ranging from 12
to 150 kDa in size, with a carbohydrate content ranging
from 30% to 67% by weight in Colletrotrichum lindemuthianum [32]. A well-dened role in cell-wall chitosan
biosynthesis has been demonstrated for CDA secreted
in the periplasm. In M. rouxii, chitin synthase rst polymerizes chitin chains, then the N-acetamido bonds are
hydrolysed by CDA [29]. In S. cerevisiae, two CDAs
are required for the biosynthesis of a chitosan-containing ascospore wall [35], resistant to stress conditions
such as glusulase or heat treatment [34,36]. In the microsporidian spore wall, chitosan could provide interactions with the other wall components and could thus
account for the extreme rigidity and resistance of the
parasite wall to chitinase treatments and to dierent
stresses such as freezing, heating and extreme pH
[9,10]. In some pathogen fungi, CDA is extracellular

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D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

(a)

(b)

Fig. 3. Sequence analysis of the ECU11_0510 protein (a) and alignment with chitin deacetylase domains from other organisms (b). (a) The full-length
protein (254 amino acids) is represented with dierent predicted elements: signal peptide (SP), secondary structure consensus with beta strand in
black and alpha helix in grey. The ECU11_0510 polysaccharide deacetylase domain encompasses the position 21150 and is included in the
pfamA01522 domain (score = 11.1, Evalue = 3.8e
05). The predicted a/b class structure ts with the crystal three-dimensional model of Bacillus
subtilis polysaccharide deacetylase (PDB: 1NY1). (b) Sequence alignment of chitin deacetylase domains from the mammalian Microsporidia
Encephalitozoon cuniculi (ECU11_0510), the insect Microsporidia Antonospora (formerly Nosema) locustae (sequence from contig_159_1742:2506,
http://jbpc.mbl.edu/Nosema/index.html, pfam: score = 56.4, Evalue = 8.3e
14), the fungi Colletotrichum lindemuthianum, Saccharomyces cerevisiae,
Mucor rouxii and the bacterium Bacillus subtilis. Identical and similar residues are highlighted in black and grey respectively. The microsporidian
domain is markedly reduced but presents a strong homology with the other proteins on its 6070 rst amino acids.

D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

87

Fig. 4. Immunogold electron microscopy of cryosections of dierent E. cuniculi developmental stages using the anti-11_0510 PAb and secondary
antibody conjugated with 5 nm colloidal gold (bars = 200 nm). The labelling (small arrows) is observed as early as the meront-to-sporont transition
in the cytoplasm (a). In dividing sporonts (b), gold particles are found at the periphery of the cell. Finally, the antibodies react with the innermost
part of the endospore of the mature spore (c). Black arrowheads highlight transversal sections of the spore wall, while the white ones show grazing
section planes. PM: plasma membrane. PDM: patches of dense material. LDM: lamellar dense material. SW: spore wall. Ex: exospore. En:
endospore. PT: polar tube.

Fig. 5. Immunogold electron microscopy of cryosections of E. cuniculi sporoblasts using the anti-11_0510 PAb and secondary reagent labelled with
5 nm colloidal gold (bars = 200 nm). In sporoblasts (a,b), the labelling (small arrows) is associated with the constructing wall (c) and concentrates in
peripheral structures (d,e). PM: plasma membrane. Ex: exospore. En: endospore.

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D. Brosson et al. / FEMS Microbiology Letters 247 (2005) 8190

and is thought to be implicated in the bypass of host-defence mechanisms during C. lindemuthianum plant invasion [32] or Aspergillus fumagitus mammalian infestation
[37].
3.3. The EcCDA protein presents two isoforms
In the E. cuniculi extract, both PAb recognized the
two ECU11_0510 isoforms at 33 and 55 kDa (Fig. 2).
Several fungi also exhibit multiple CDA glycoproteins:
two, three, four and up to ve isoforms were described
in Mucor rouxii [31], Metarhizium anisopliae [38], Rhizopus nigricans [39] and Uromyces viciae-fabae [40], respectively. The E. cuniculi genome sequence predicts only
few genes involved in protein glycosylation, all of them
being related to O-mannosylation [7]. Sixteen serine and
eleven threonine residues are potential O-glycosylation
sites in EcCDA. While O-mannoproteins were recently
detected in the spore wall of E. cuniculi and in the
SDSPAGE 55-kDa region (Metenier, G., Mazet, M.
and Taupin, V., personal communication), concanavalin-A labelling did not co-localize with the
ECU11_0510 protein on 2-D gels (not shown).

ciated with membranes of this structure that is lled

with an electron-dense material similar to that of the
endospore. Fig. 5(e) suggests that this material is owing
between the plasma membrane and the future exospore.
In dierent Microsporidia such as Pleistophora sp.,
Stempellia sp. and Ameson pulvis, a similar membranous
whorl termed scindosome or paramural body has
been observed at the furrowing sites during division
stage of the parasite [13,41].
Since the sporoblast decreases in size as its dierentiation proceeds, the E. cuniculi paramural body could be
interpreted as an endocytic recuperation of membrane/
endospore material. Alternatively, chitin might be synthesized and deacetylated within the paramural body before being incorporated to the wall by an exocytosis-like
process. As a result, this structure would be the equivalent of the fungal chitosome, an organelle which is specialized for chitin biosynthesis [42,43]. The chitosome
hypothesis should be tested through further studies on
the expression and location of microsporidian chitin
synthase (ECU01_1390).

Acknowledgments
3.4. EcCDA is located at the plasma membrane
endospore interface of the parasite
Immunocytochemical experiments at the ultrastructural level with the anti-11_0510 PAb, revealed a slight
gold labelling of the cytoplasm of stages representative
of the meront-to-sporont transition (Fig. 4(a)). Such
cells are indeed still tightly attached to the parasitophorous vacuole membrane by one side, and their engagement in early sporogony is marked by patches of
dense material abutting on the plasma membrane. As
sporogony progresses, the labelling tends to accumulate
at the periphery of the parasitic cells. In longitudinal sections of dividing sporonts (Fig. 4(b)), gold particles are
scattered just beneath the lamellar dense material deposit on the plasma membrane. In mature spores (Fig. 4(c)),
gold particles are mainly seen at the plasma membrane
endospore interface. In sporoblasts, the EcCDA is also
present in the inner part of the cell wall tightly associated with the plasma membrane (Fig. 5(a)(c)). This
location is consistent with biochemical and sequence
data, and suggests a role in cell-wall formation. Late
sporogony is characterized by the thickening of the chitin-containing endospore [12], but the mechanism of chitin deposition is unknown. Since the EcCDA enzyme is
regularly distributed at the plasma membrane throughout sporogony, chitin may be produced all around the
cell.
Late sporoblasts frequently harbour a membranerich structure (300 200250 nm) which reacts with
EcCDA-specic PAb (Fig. 5(a), (b), (d) and (e)), and
which opens to the wall. Gold particles are clearly asso-

D. Brosson was supported by a grant from Ministe`re de lEducation, de la Recherche et de la Technologie. We thank G. Metenier for helpful discussion
and critical reading of this manuscript. We also thank
B. Chebance and I. Wawrzyniak for E. cuniculi cell culture and R. Guerry and A. Voldoire for technical advice.

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