Download Role of the posterior vacuole in Spraguea lophii (Microsporidia

FOLIA PARASITOLOGICA 52: 111–117, 2005
Role of the posterior vacuole in Spraguea lophii (Microsporidia)
spore hatching
Ann M. Findley1, Earl H. Weidner2, Kevin R. Carman2, Zhimin Xu3 and J. Samuel Godbar3
1
Department of Biology, University of Louisiana at Monroe, Monroe, Louisiana 71209, USA;
2
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA;
3
Department of Food Sciences & Human Ecology, Louisiana State University, Baton Rouge, Louisiana 70803, USA
Key words: catalase, peroxisomal vacuole, microsporidian spore extrusion apparatus
Abstract. Microsporidia constitute a large group of obligate intracellular protozoan parasites that inject themselves into host
cells via the extrusion apparatus of the infective spore stage. Although the injection process is poorly understood, its energy
source is thought to reside in the posterior vacuole that swells significantly during spore firing. Here we report the presence and
localisation of the key peroxisomal enzymes catalase and acyl-CoA oxidase (ACOX) within the posterior vacuole of Spraguea
lophii (Doflein, 1898) spores. Western blot analyses show that these enzymes discharge out of the spore and end up in the medium external to the extruded sporoplasms. The presence of a catalase enzyme system in the Microsporidia was first made evident by the detection of significant levels of molecular oxygen in the medium containing discharging spores in the presence of
hydrogen peroxide. Catalase was visualised in inactive, activated, and discharged spores using alkaline diaminobenzidine (DAB)
on glutaraldehyde-fixed cells. The position of these enzymes within the extrusion apparatus before and during spore discharge
support the Lom and Vávra model that postulates discharge occurs by an eversion process. In addition to these enzymes, spores
of S. lophii contain another characteristic peroxisomal component, the very long chain fatty acid (VLCFA) nervonic acid. A sizeable decrease in nervonic acid levels occurs during and after spore discharge. These data indicate that nervonic acid is discharged
from the spore into the external medium during firing along with the catalase and ACOX enzymes.
Peroxisomes are found in virtually all mitochondriabearing eukaryotes that have long-term associations
with oxygen, hydrogen peroxide detoxification, and
oxidation of very long chain fatty acids (Mullen and
Trelease 1996). We report a peroxisomal-like organelle
in a primitively prescribed amitochondriate microsporidian, Spraguea lophii (Doflein, 1898). Although
best known as intracellular parasites of fish and arthropods, microsporidians are eukaryotes that have achieved
a wide distribution in nature (Canning and Lom 1986).
Microsporidians are noteworthy because of their invasive spore stage that functionally resembles a missile
cell that is uniquely adapted for injecting its infective
contents into a target host cell. The firing mechanism
includes a coiled protein filament surrounded by membrane pleats and a posterior vacuole (Weidner et al.
1995). During spore activation, the posterior vacuole
swells significantly followed by an explosive evertive
discharge of the tube through a spore aperture with millisecond velocity. It is through the invasion tube that the
spore contents (sporoplasm) are ejected into a newlyformed membranous sac at the tube tip. After discharge,
the everted contents of the extrusion apparatus end up
exterior to the sporoplasm cells. We report major molecular markers for peroxisomes within the microsporidian spore extrusion apparatus: very long chain fatty acids (nervonic acid), acyl-CoA oxidase (ACOX), cata-
lase, and the most interactive of the peroxisomal proteins (peroxin) Pex19.
MATERIALS AND METHODS
Source of S. lophii spores from Lophius americanus. Microsporidian cysts containing S. lophii spores were recovered
from the infected neurons of the anglerfish, Lophius americanus (Valenciennes, 1837). All of the infected animals used
in this study were provided by the Marine Biological Laboratory Aquatics Facilities at Woods Hole, MA. The cysts were
dissociated with a glass homogenizer and the spores were
purified through repetitive centrifugation/wash cycles. Purified spores were stored in distilled water (with a trace of calcium) at 4°C. Spores were prepared for activation and hatching as described previously (Weidner et al. 1995). It should be
noted that the classification scheme for the group of microsporidians that infect the anglerfish is currently under revision
and that the species designation of the organism used in this
study may soon be changed (Freeman et al. 2004).
Oxygen generation as a measure of catalase. Oxygen
saturation levels were monitored continuously in the medium
bearing discharging S. lophii spores (in Hepes buffer, pH 7)
and were measured with a Clarkstyle microelectrode (Model
This paper was presented at the NATO Advanced Research Workshop “Emergent Pathogens in the 21st Century: First United Workshop on Microsporidia from Invertebrate and Vertebrate Hosts”, held
in České Budějovice, Czech Republic, July 12–15, 2004.
Address for correspondence: A.M. Findley, Department of Biology, University of Louisiana at Monroe, Monroe, LA 71209, USA.
Phone: ++1 318 342 1817; Fax: ++1 318 342 3312; E-mail: [email protected]
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7376C, Diamond General, Ann Arbor, MI). Since catalase was
believed to be extruded directly into the external medium,
H2O2 (0.01%) was added to this medium with spore activation.
In the control treatment, no H2O2 was added to the medium
bearing firing spores.
ACOX measurement. ACOX activity was determined
colorimetrically at 550 nm (Wako Chemical, Richmond, VA).
Briefly, when substrate is oxidized by ACOX, any H2O2 produced reacts with added peroxidase to cause the oxidative
condensation of 3-methyl-N-ethyl-N-aniline with 4-aminoantipyrine. The formation of a purple-coloured product can be
followed spectrophotometrically. A standard curve was determined with known ACOX levels present. Experimental preparations were measured in the absence of kit ACOX and consisted of different concentrations of hatched spores (bearing
their own resident ACOX enzyme).
Very long chain fatty acids. During esterification, samples were dissolved in 5 ml of hexane to which a C17.0 fatty
acid was added as an internal standard. Following the addition
of 2 ml BCl3-methanol (12% w/w), sample mixtures were
incubated at 60°C for 10 min after which time 1 ml water was
added. The hexane supernatant layer was separated by centrifugation and filtered through Na2SO4 (anhydrous) before
transfer to a GC vial. Samples were analysed for fatty acid
composition utilizing the GC-FID (HP5890, Hewlett-Packard,
Avondale, PA) equipped with a SP2380 capillary column
(Supelco, Bellofonte, PA). The GC oven was held at 50°C for
2 min, increased to 250°C, and held there for 15 min. The
temperature of the injection port and the detector was 250°C.
The split ratio was 1/100.
Western blot analysis. Monoclonal antibodies to peroxin
(Pex19) (BD Transduction Laboratories, Lexington, KY) and
catalase (clone CAT-505, Sigma, St. Louis, MO), and polyclonal antibodies to ACOX (gifts of R. Rachubinski and S.
Alexson) were used in this study. Secondary antibodies coupled to alkaline phosphatase were purchased from Sigma.
Standard protocols were followed for Western blot analysis.
Diaminobenzidine (DAB) probe for catalase and standard electron microscopy. Diaminobenzidine (DAB) was
used to visualise catalase activity in microsporidian spores.
The protocol employed included prefixing cells with 2.5%
glutaraldehyde and subsequent incubation in Tris buffer (pH
10.5) in the presence of DAB and elevated H2O2 levels. These
conditions eliminate any resident peroxidase activity (Angermuller and Fahimi 1981).
To document the swelling of the posterior vacuole extrusion apparatus during spore germination, spores were activated and fixed quickly before they fired. This procedure enabled the capturing of images in which the vacuole continues
to swell up to the point of spore firing. Standard electron microscopy protocols were employed.
RESULTS
Catalase is the centrepiece enzyme of the eukaryotic
peroxisome (Purdue and Lazarow 1996, 2001). Its presence in S. lophii was first indicated by the production of
significant levels of molecular oxygen in medium containing discharging spores (Fig. 1a). The oxygen surge
resulted when hydrogen peroxide was added either
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slightly before or during spore discharge. The amount of
oxygen generated was proportional to the level of spore
firing and the amount of H2O2 substrate added to the
medium containing primed spore material. In the absence of hydrogen peroxide, activated spores yielded no
detectable oxygen and spore hatching was greatly reduced. The enzyme ACOX is always found in association with peroxisomes (Van Veldhoven et al. 1991,
Wanders et al. 2001). Colorimetric determination of
ACOX levels in hatching spores of S. lophii revealed
significant amounts of enzymatic activity present with
the amount of activity dependent upon spore sample
concentration, i.e., a tenfold increase in ACOX activity
was observed with a tenfold increase in spore sample
material (Fig. 1b, M1 vs. M2). Nervonic acid (C24:1)
was the only detectable very long chain fatty acid
(VLCFA) discharged along with the catalase and
ACOX enzymes into the medium external to the discharged sporoplasm cells. To determine whether there
was β-oxidation of nervonic acid in the medium, aliquots were examined 5, 30 and 60 min after spore discharge. The results showed a marked decrease in the
nervonic acid profile of the medium during the first
thirty minutes of incubation (Fig. 1c). The data presented in Fig. 1 provide a representative series of several experiments performed for each parameter measured (O2 generation, ACOX levels, and nervonic acid
concentrations). Statistical treatment of these data was
deemed impracticable due to large variations in percent
spore firing between each experiment and the difficulty
associated with counting spores that clump together
upon discharge.
Western blot analyses were used to identify microsporidian ACOX, catalase, and Pex19. Antibodies directed against ACOX were tested on mouse liver peroxisomes and discharged S. lophii spore material. A
single 72-kDa peptide was found in both samples (Fig.
2a). The ACOX protein was present in the supernatant
of fired spores rather than in the sporoplasm-containing
pellet. Two forms of catalase were identified from extruded spores. Bands ascribed to the presence of catalase A (CATA, 72 kDa) and catalase B (CATB, 60 kDa)
proteins correlated with those observed in Neurospora
crassa and the CATB protein described in mouse liver
peroxisomes (Fig. 2b). As was the case for ACOX, catalase activity was absent in the sporoplasm pellet of S.
lophii but was located exclusively instead in the external
medium bearing the everted contents of the spore extrusion apparatus. Finally, the presence of Pex19 was ascertained since this peptide represents one of the most
interactive peroxins relative to other peroxin proteins (at
least 14) and hence is considered to be a good indicator
of peroxisomal activity (Gotte et al. 1998, Sacksteder et
al. 2000). Monoclonal antibodies to mouse liver Pex19
evidenced a single band of 40 kDa. The microsporidian
sample yielded a single band for Pex19 but with a
higher molecular weight (50–55 kDa, Fig. 2c).
Findley et al.: Microsporidian spore hatching
Fig. 2. Western blots of peroxisome enzymes. a – ACOX.
Lane a1 shows a 72-kDa ACOX band in the supernatant of
discharging spore. Lane a2 shows that the Spraguea lophii
sporoplasm pellet is free of ACOX. Lane a3 contains the
ACOX band from mouse liver peroxisomes for comparison.
b – Catalase. Lane b1 shows 72-kDa CATA and 60-kDa
CATB bands from S. lophii. Lanes b2 and b3 show known
catalase bands from Neurospora crassa (72 kDa and 60 kDa)
and mouse liver peroxisomes (60 kDa), respectively. c – Peroxin 19. Antibody is directed against peroxin (Pex19). In c1,
mouse liver peroxisomal Pex19 gives a single 40-kDa band.
C2 demonstrates a clear band against Pex19 from S. lophii,
however, the peptide is believed to be between 50–55 kDa.
Fig. 1. Peroxisomal activity in Spraguea lophii. a – Oxygen
generation with addition of 0.01% H2O2 to medium containing
106 hatching spores (closed circles); without H2O2 substrate
(open circles), no O2 detection was recorded. b – Colorimetric
measurement of ACOX. S0 was a standard recorded without
ACOX in medium containing hatching spores; S1 and S2 are
external standards with 10 and 20 units of ACOX present in
medium. M1 and M2 are experimental treatments with the
only ACOX present coming from the spore material. M2
represents a 10× increase in spore sample. Note that the
ACOX colorimetric measurement indicates a concomitant 10×
increase in enzyme activity with increased spore concentration. c – Nervonic acid (C24:1) is a very long chain fatty acid
found in the medium with discharged S. lophii spores. In a
time course experiment following spore germination, the results show a sharp decline (between 0 and 30 min) in nervonic
acid indicating β-oxidation of this fatty acid.
Catalase was selectively visualised with diaminobenzidine (Angermuller and Fahimi 1981, Yokota et al.
1987). In the presence of catalase, DAB reacts with
H2O2 to form a precipitate that was confined to the microsporidian spore posterior vacuole (Figs. 3b, c). As
the invasion tube emerges from the firing spore, the
DAB reaction is associated first with the inner surface
and then subsequently along the outer surface of the
discharged tube (Weidner and Findley 2002, 2003).
After spore discharge, no detectable DAB activity was
found in either the spore ghosts or the extruded sporoplasms. Control spores that were primed for hatching
but for which the hydrogen peroxide substrate was not
provided were negative for the DAB reaction product;
however, the posterior vacuole was clearly increased in
size when compared to unactivated spores (Fig. 3a).
Before activation, S. lophii spores have an extrusion
apparatus that bears a posterior vacuole that is much
reduced in size. This vacuole is osmiophilic and does
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Fig. 3. Catalase visualised by diaminobenzidine. a – Spraguea lophii spores do not react to DAB in the absence of H2O2 in the
medium, pH 10.5. b, c – The reaction of spores with DAB in the presence of H2O2. Spore cells were prefixed with glutaraldehyde
to eliminate any resident peroxidase activity. Arrows show that all catalase activity is confined to the extrusion apparatus posterior vacuole. Scale bars = 0.5 µm.
react with the lipid probe, Nile Red (not shown). During
spore activation, the posterior vacuole grows significantly and rapidly until it occupies over half the spore
volume (Figs. 4a, b). Ultimately, the spore aperture collapses and the extrusion apparatus everts and the sporoplasm is sent out of the spore with millisecond velocity
(Fig. 4c). As the sporoplasm passes out of the spore, the
spore compartment collapses and the completely discharged spore appears flattened.
Based upon the identification and localisation of key
enzyme systems (catalase, ACOX) and metabolites (O2,
VLCFA) before, during, and following spore discharge,
we propose a composite model for spore germination
(as illustrated in Fig. 5). The microsporidian spore extrusion apparatus consists of the spore aperture, polar
filament protein coil, polaroplast membrane system, and
posterior swelling vacuole. Previous studies have shown
that when the extrusion apparatus discharges, the polar
filament coil (shown in blue) emerges through the spore
aperture instantaneously along with the polaroplast
membrane (shown in red) and that these form the invasion tube by an eversion process (Lom and Vávra 1963,
Weidner et al. 1995). In the present model of spore
hatching, the extrusion apparatus membrane is exteriorized and becomes the outer envelope of the discharged
sporoplasm cell. As the extrusion apparatus completely
everts, the contents of the posterior vacuole (catalase,
ACOX, VLCFA; represented by black stars) are discharged into the external medium. The sporoplasm (nucleus and cytoplasm; labeled c and represented in yellow) enters into the compartment formed from the
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everted polaroplast membrane. The original plasma
membrane is left behind on the spore wall.
DISCUSSION
Despite the importance of the Microsporidia as the
etiological agents of disease in a wide variety of invertebrate and vertebrate hosts, the specifics of the spore
hatching process are, as of yet, poorly understood. Although the sudden swelling of the posterior vacuole has
been implicated in playing a pivotal role in the force
generation necessary for spore germination (Lom and
Vávra 1963, Weidner et al. 1995), the details of the
mechanism by which this might occur have remained
undetermined. Interest in the study of posterior vacuole
dynamics during spore discharge stems from the demonstrated efficacy of small amounts of H2O2 in in vitro
hatching protocols of spore smears and the observation
of O2 generated concomitant with this H2O2 addition
(Weidner and Findley 2002, 2003). Oxygen evolution
was shown to be proportional to spore firing efficiency
and the amount of H2O2 added to spores primed for
hatching. In as much as H2O2 serves as the substrate for
the antioxidant enzyme catalase, we sought to demonstrate the presence of this enzyme and to localise its
position within the microsporidian spore prior to and
during spore germination. The DAB reaction product
indicates the presence of catalase is initially confined
within the posterior vacuole, subsequently moves to the
extruding polar tube during the hatching process, appears on the outside of the discharged tube, and finally
Findley et al.: Microsporidian spore hatching
Fig. 4. Electron micrographs of activated Spraguea lophii
spores. a, b – A significant increase is seen in the volume of
the posterior vacuole following spore activation. c – Additional increase in vacuole size results as the extrusion apparatus is discharged. It is presumably within the extrusion apparatus where the catalase, nervonic acid, and other components of
the peroxisome are located. The volumetric shift within the
vacuole is likely due to β-oxidation within the activated spore
peroxisome and accompanying water production. Scale bars =
0.5 µm.
diffuses into the surrounding medium that bathes the
discharged sporoplasms. These results have been confirmed via western blot analysis of a monoclonal antibody to catalase tested against hatched sporoplasm material (negative for catalase) and the surrounding medium contents (positive for catalase). Furthermore, we
have recently isolated catalase crystals from S. lophii
spore material and the purification and characterisation
of these crystals is currently in progress (Weidner and
Findley 2002).
Although the specifics of the mechanism by which
catalase works in the posterior vacuole remain unclear,
it may be associated with the ß-oxidation of very long
chain fatty acids. VLCFA have been reported in significant amounts within Spraguea lophii spores (El Alaqui
et al. 2001). Nervonic acid is a characteristic peroxisomal component (Sandhir et al. 1998), and the data
indicate that it is discharged from the spore upon hatching. The decrease in nervonic acid levels observed following spore hatching and the presence of the catalase
and ACOX enzymes in the external medium surrounding discharged spores are consistent with ß-oxidation
activity associated with the posterior vacuole contents
and its probable identification as a peroxisomal-like
organelle. Acyl-CoA oxidase is an essential enzyme of
lipid catabolism and its action produces hydrogen peroxide. The co-location of catalase and ACOX within the
same cytoplasmic compartment, namely the posterior
vacuole, provides an efficient means for the conversion
of the H2O2 produced during ß-oxidation of VLCFA to
H2O and O2. Consequently, the oxidation of long chain
fatty acids and its resultant production of significant
amounts of water and molecular oxygen may lead to the
observed rapid swelling of the posterior vacuole. This
volumetric increase in the posterior vacuole ultimately
causes the spore aperture to be breached and begins the
evertive discharge of the spore.
In the amitochondriate Microsporidia, the peroxisome becomes the only logical candidate for VLCFA
assembly and ß-oxidation. The data presented are consistent with the identification of the posterior vacuole of
the microsporidian spore as a presumptive (primitive or
extremely specialised) peroxisomal organelle. Western
blots of external media samples collected from hatching
spores have been probed with antisera raised against
mammalian peroxisomal matrix proteins and have successfully identified a number of peptides with peroxisomal-leading sequences (Fransen and Weidner, unpublished data).
Peroxisomes are extremely adaptable organelles that
exhibit considerable functional plasticity performing
disparate roles in different organisms (Titorenko and
Rachubinski 2001, 2004). Although best known for
their role in lipid catabolism and H2O2 detoxification,
recent evidence also suggests a prominent role for this
organelle in the intracellular signaling processes operative during development. In this context, peroxisomes
may function as an organizing and coordination centre
providing critical information that affects differentiation
and morphogenesis events within cells (Titorenko and
Rachubinski 2004). Derived from the endoplasmic reticulum of cells, the peroxisome has been shown to be
structurally diverse as well. For example, in yeast the
overexpression of certain Pex genes that code for the
peroxin proteins needed for peroxisome assembly can
result in a small number of greatly enlarged peroxi-
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Fig. 5. Model of microsporidian spore discharge. Probable sequence of firing events during the eversion of the polar filament
which represents a modification of the Lom and Vávra (1963) model. C indicates the cytoplasmic contents of the posterior vacuole that are discharged into the surrounding medium. Blue – polar filament protein; red – membrane of extrusion apparatus; yellow – spore cell cytoplasm; *** – contents of posterior vacuole, the peroxisomal proteins, that are expelled during spore hatching.
somes in affected cells (Titorenko and Rachubinski
2001, 2004).
The body of data presented herein point to the posterior vacuole of the microsporidian spore as a peroxisome-like organelle. Biochemical, cytochemical, and
western blot analyses have identified and localised several key peroxisomal components, namely catalase,
ACOX, Pex19, and nervonic acid oxidation, as being
associated with the posterior vacuole of S. lophii spores.
Following spore hatching, all of these components, and
O2 generation, are found in association with the external
medium that bathes the sporoplasm cells. These results
support the Lom and Vávra (1963) model of spore discharge. In this model, the posterior vacuole swells upon
spore activation, the extrusion apparatus everts, and a
membranous sac forms at the tip of the discharged tube
to accommodate the nascent spore cell, or sporoplasm.
Additionally, this model implies that the membrane of
the extruded sporoplasm may be derived from the extrusion apparatus and may therefore be reversed. Indeed,
preliminary studies with cytoplasmic protein probes
indicate that in discharged sporoplasms, the membrane
orientation is reversed as proposed (De Giorgis and
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Weidner, unpublished data). Comparative studies of
catalase activity and posterior vacuole dynamics in
other microsporidian species are currently underway to
adjudicate whether the spore firing mechanism proposed
here is in widespread use throughout the Microsporidia.
Additionally, whether catalase activity and posterior
vacuole dynamics are solely responsible for spore firing
in S. lophii has yet to be established. The role of increased osmotic potential within germinating spores as a
result of trehalose conversion to glucose has also been
implicated as a possible spore firing mechanism in
aquatic microsporidian species (Undeen and Vander
Meer 1999). Further studies are warranted to discern the
relative importance of these mechanisms in the hatching
process.
Acknowledgements. This work was supported in part by the
Howard Hughes Undergraduate Biological Sciences Education
Program at the University of Louisiana at Monroe (grant no.
52002691) and by NIH grant P20RR16456 from the BRIN
Program of the National Center for Research Resources. The
authors wish to thank D. Burks (Louisiana State University)
for his assistance in the preparation of the line-drawn figures.
Findley et al.: Microsporidian spore hatching
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Received 3 January 2005
Accepted 18 March 2005
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