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Morphological characterization of Cryptosporidium parvum life-cycle stages in an in vitro model system

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Cryptosporidium parvum is a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the potential to cause severe enteric disease. We describe the complete life cycle of C. parvum in an in vitro system. Infected cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well as determining their time of occurrence after inoculation. Numerous stages of C. parvum and their behaviour have been visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host interactions and the effect of C. parvum on host cells were also visualized. An improved understanding of the parasite's biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.
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Morphological characterization of Cryptosporidium parvum
life-cycle stages in an in vitro model system
H. BO RO WS KI
1
,R.C.A.THOMPSON
1
*, T. AR MSTR ON G
1
and P. L. CLODE
2
1
WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections,Veterinary and Biomedical Sciences,
Murdoch University,South Street,Murdoch,WA 6150,Australia
2
Centre for Microscopy,Characterisation and Analysis,The University of Western Australia,35 Stirling Hwy,Crawley,
WA 6009,Australia
(Received 30 January 2009; revised 25 May, 15 June and 19 June 2009; accepted 22 June 2009; first published online 20 August 2009)
SUMMARY
Cryptosporidium parvum is a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the
potential to cause severe enteric disease. We describe the complete life cycle of C. parvum in an in vitro system. Infected
cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional
data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well
as determining their time of occurrence after inoculation. Numerous stages of C. parvum and their behaviour have been
visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host
interactions and the effect of C. parvum on host cells were also visualized. An improved understanding of the parasite’s
biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.
Key words: Cryptosporidium parvum, morphology, host cell interaction, phylogenetic affinity, gregarines, electron
microscopy.
INTRODUCTION
Cryptosporidium is a protozoan enteric parasite of
humans and other vertebrates (Fayer et al. 1997).
Numerous Cryptosporidium species have been de-
scribed (Smith et al. 2005) most of which are specific
to their vertebrate host. The species C. parvum is of
medical and economic relevance as it affects both
humans and cattle with its primary site of infection
being the gastrointestinal tract. It affects the epi-
thelial lining of the ileum, resulting in self-limiting
diarrhoea in immunocompetent individuals or in
life-threatening diarrhoeal diseases in immunocom-
promised individuals.
Belonging to the phylum of apicomplexan para-
sites, Cryptosporidium shares common life-cycle
features and morphological characteristics with
other members of this phylum (Tetley et al. 1998).
Initially, Cryptosporidium was categorized as a cocci-
dian parasite (Levine, 1988). However, more recent
studies show that Cryptosporidium lacks key mor-
phological characteristics of coccidians and is insen-
sitive to anti-coccidial agents (O’Donoghue, 1995 ;
Fayer et al. 1997; Carreno et al. 1999). Further
phylogenomic analysis has since revealed that
Cryptosporidium is most closely related to gregarines
(Barta and Thompson, 2006). Cryptosporidium shares
many features in common with gregarines, including
an extracytoplasmic location and connection to the
host cell via a myzocytosis-like feeding mechanism
(Barta and Thompson, 2006). The primary differ-
ence between these two groups is that Crypto-
sporidium induces the host cell to overlay it with the
host cell apical membrane (Barta and Thompson,
2006; Butaeva et al. 2006). The parasite appears on
the surface of cells, residing in a parasitophorous
vacuole (PV) between the cytoplasmic membrane
and the apical membrane (Huang et al. 2004).
Critically, the mechanisms of Cryptosporidium
pathogenesis are not fully understood, but both
parasite stimuli as well as host immune responses are
thought to play critical roles (Barta and Thompson,
2006). The life cycle and the mechanisms of infection
by Cryptosporidium have recently been reviewed
in detail by Smith et al. (2005) and Borowski et al.
(2008). Importantly, previous studies by Hijjawi
et al. (2001, 2004) described the life cycle of C. par-
vum in vitro, using light microscopy while more
recent studies by Valigurova et al. (2008) described
the morphology of various life-cycle stages of 2 dif-
ferent Cryptosporidium species from mice and toads
in vivo using electron microscopy.
The aim of this study was to expand on this earlier
work and to gain a better understanding of the
biology and relationship with host cells of the
* Corresponding author: WHO Collaborating Centre
for the Molecular Epidemiology of Parasitic Infections,
Veterinary and Biomedical Sciences, Murdoch University,
South Street, Murdoch, WA 6150, Australia. Tel : +(08)
9360 2466. Fax: +(08) 9360 6285. E-mail: a.thompson@
murdoch.edu.au
13
Parasitology (2010), 137, 13–26. fCambridge University Press 2009
doi:10.1017/S0031182009990837 Printed in the United Kingdom
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economically and medically important species,
C. parvum. In this study the human ileocecal epi-
thelial cell line HCT-8 was used as an in vitro model
to monitor the developmental process of C. parvum
and to study the effects upon target cells. Crypto-
sporidium was observed to proliferate in our culture
system for 5 days. Hence, infected cells were moni-
tored for this period, with data obtained using
scanning (SEM) and transmission (TEM) electron
microscopy. From this, a more complete life cycle
of C. parvum has been visualized and the behaviour
and morphological characteristics of numerous life-
cycle stages described for the first time with the aid
of SEM.
MATERIALS AND METHODS
Cell culture
The C. parvum cattle isolate used during this
study was originally obtained from the Institute of
Parasitology, University of Zurich. Oocysts were
subsequently passaged through, and purified from,
infected ARC/Swiss mice as described by Meloni
and Thompson (1996). For routine passaging,
HCT-8 cells were cultured in RPMI medium with
2gL
x
1
sodium bicarbonate, 0.3g L
x
1
L-glutamine,
3.574 g L
x
1
HEPES buffer (15 mmol L
x
1
) and 10%
fetal calf serum (FCS) at pH 7.4, 37 xC with 5 % CO
2
.
Pre-treatment of oocysts
C. parvum oocysts were bleached with 200 mlof
household bleach in 10 ml of water for 30 min at
room temperature (RT). Sterilized oocysts were
inoculated into excystation medium (0.5 % trypsin,
pH of 2.5) for 30 min at 37 xC. Excysted oocysts were
resuspended in maintenance medium consisting of
the RPMI medium described above plus 3 g L
x
1
sodium bicarbonate, 0.2gL
x
1
bovine salt, 1 g L
x
1
glucose, 250 mgL
x
1
folic acid, 1 mg L
x
1
4 amino
benzoic acid, 500 mgL
x
1
calcium pentothenate,
8.75 mg L
x
1
ascorbic acid and 1 % FCS.
Cell line infection and cell-free culture
Twenty-four h prior to an infection of cells with
C. parvum, HCT-8 cells were plated onto thermonox
cover slips in 24-well plates. Each cell was then
infected with C. parvum pre-treated oocysts
(15 000 per cm
2
) in 1 ml of maintenance medium and
maintained at 37 xC with 5 % CO
2
. Infected cultures
were sampled and processed for microscopy at 6 h,
7 h, 24 h, 48 h, 72 h, 96 h and 120 h post-inoculation.
Sample preparation for electron microscopy
Cover-slips with adherent cells were fixed in
2.5% glutaraldehyde in 1rPBS. Additionally, to
investigate extracellular C. parvum stages present
within the supernatant of infected cells, medium
from cells was aspirated and added to an equal
volume of 5% glutaraldehyde in 2rPBS. Cellular
material within this supernatant was subsequently
attached to poly-L-lysine coated glass cover-slips
for SEM investigation by applying several drops
of concentrated supernatant and incubating for
20 min.
All samples were post-fixed in 1 % OsO
4
in PBS,
and then dehydrated in a graded series of ethanols
using a Pelco Biowave Microwave Processor.
Samples destined for SEM were then critical-point
dried, mounted on stubs with carbon tabs, and
coated with 3 nm platinum for high-resolution
imaging. Samples destined for TEM were infil-
trated and embedded in Spurr’s Resin. Thermonox
cover-slips were removed under liquid nitrogen,
and samples re-embedded. Sections, approximately
100 nm thick, were cut on a diamond knife and
mounted on copper grids.
Imaging
SEM images were acquired at 3 kV using the in
lens secondary electron detector, on a Zeiss 1555VP
field emission SEM. TEM sections were viewed
unstained at 120 kV using a JEOL 2100 TEM.
Images were digitally acquired with a Gatan SC1000
ORIUS digital camera.
RESULTS AND DISCUSSION
All recognized C.parvum life-cycle stages (Table 1)
were observed on the surface of epithelial cells or in
the supernatant. Small trophozoites (<1mm) were
observed as early as 6 h post-inoculation with well-
distinguishable meronts I and free merozoites type I
being observed after 24 h. This implies that oocyst
excystation and sporozoite invasion must have
occurred immediately post-inoculation. Consistent
with this, previous studies by Forney et al. (1999),
observed sporozoites invading host cells as early
as 5 min post-inoculation at the point of sporozoite
emergence from the oocyst. Subsequent infection
increased over the next 2–3 days as a result of ongoing
oocyst excystation, and the ability of C. parvum
to replicate asexually. After 2 days post-infection,
only inoculated oocysts, sporozoites, trophozoites,
meronts I and merozoites type I were observed in
culture. After 3 days, meronts II as well as mero-
zoites type II were seen, while gametocytes were
not observed until 4 days post-inoculation.
This timeline of Cryptosporidium development
correlates with previous light microscopic data from
in vitro cultures of C. parvum (Hijjawi et al. 2001,
2002, 2004), as well as electron microscopy studies
of Cryptosporidium sp. ‘ toad’ and C. muris from
experimentally infected toads and mice respectively
H. Borowski and others 14
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(Valigurova et al. 2008). This consistency with an
in vivo system confirms the validity of our in vitro
model system of C. parvum infection.
In addition to the widely accepted life-cycle stages,
we observed stages that are not commonly reported
or known to date (Table 1). Such stages include
trophozoites that formed within parent stages with-
out host cell invasion, and extracellular accumu-
lations of trophozoites.
Oocyst excystation and sporozoite host cell invasion
Following inoculation, intact C. parvum oocysts
were only ever found in the supernatant and these
were ovular, 5 mmr7mm in size and possessed a
smooth surface (Fig. 1A). This correlates with pre-
vious findings of Hijjawi et al. (2001). On the surface
of these oocysts a cleft was visible and presumably
it is along this cleft that the oocyst opens to release
sporozoites during the excystation process. Excysted
oocysts were observed adhered to cells after 24 h but
not before. It is known that oocysts possess surface
lectins that are thought to hinder their adherence to
host tissue in vivo until the target tissue is reached
where they then mediate attachment to host cells
(Kuznar and Elimelech, 2006). In the present study,
oocysts were directly applied to target cells but did
not attach immediately. Perhaps without passage
through the gastrointestinal tract, oocysts do not
begin to express the surface lectins needed for host
cell adherence until after extended exposure to host
cells or simply until a certain progression of time
after excystation stimuli are initiated. The outer
membrane of excysted oocysts appeared rough
(Fig. 1B). This is probably due to membrane per-
foration during the excystation processes.
Sporozoites observed in this study measured about
5mmr0.5mm and showed well-defined apical ends
(Fig. 1B–D). The hatching sporozoite shown in
Fig. 1B as well as the sporozoite on the surface of
the host cell in Fig. 1D, both showed an enlarged
posterior region and a well-defined apical region,
which appeared thin and elongated. Sporozoite
apical organelle discharge is known to mediate host
cell contact and according to previous findings
already occurs during excystation (Snelling et al.
2007). As the sporozoites shown in Fig. 1B and
D appear to be making host cell contact, apical or-
ganelle discharge of molecules, which are discharged
in the presence of host cells, might account for the
occurrence of this thin and elongated apical region.
In contrast, sporozoites isolated from supernatant
did not display this apical elongation and their shape
remained largely regular along their entire length
(Fig. 1C). Sporozoites were only observed up to
48 h post-inoculation. As sporozoites are known to
invade host cells within 5 min after excystation
(Forney et al. 1999) it can be assumed that after
Table 1. Distinguishing characteristics of Cryptosporidium life-cycle stages
Time
(h) Stage Size Morphological Feature Fig.
0 Oocyst 5r7mm Ovular, smooth surface with cleft for
sporozoite release
1A
>24 Excysted oocyst 5r7mm Perforated surface 1B
>3 Sporozoite 5r0.5mm Rough surface, pointed apical region
(elongated when in proximity to host cells),
rounded posterior region
1B–D
>6 Early trophozoite <1mm Smooth surface formed by the host cell
apical membrane, hood like shape
1E
>24 Trophozoite 2.5mm Epicellular, smooth surface, electron dense
band, feeder-organelle, PV, cytoplasmic
granulation, hood like shape
2A–G
>24 Trophozoite
clusters
Merging of apical membranes
engulfing individual parasites
2D–G
>24 Meronts I 1.5mm Epicellular, smooth surface 5A,B
>24 Merozoites
Type I
0.4r1mm Rod like shape, pointed
apical region, rough surface
5E–H
>72 Meronts II 3.5mm Epicellular, smooth thick membrane 6A
>72 Merozoites
Type II
0.5–1 mm Round, rough surface 6A–C
>96 Microgamont 2r2mm Extracellular, densely packed with
microgametes
8A–C
>96 Microgamete 0.1mm Spherical, rough surface 8A–C
>96 Macrogamont 4r5mm Extracellular, ovular, rough surface 7A–C
48 Extracellular
trophozoite
2mm Forms within parent stage, rough surface 9A
96 Extracellular
meront
<2mm Round, extracellular
accumulation of trophozoites
9B
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48 h all oocysts have excysted and that no new
sporozoites are produced. Invasion of host cells by
sporozoites is shown in Fig. 1E. Invading sporozoites
eventually become completely encapsulated by the
host cell apical membrane, which initially appeared
as a hood-like structure at this early invasion stage
(Fig. 1E). A similar process has also been described
in the in vivo toad system by Valigurova et al. (2008).
As part of the infection process, sporozoites trans-
form into the trophozoite stage and undergo mer-
ogony, which leads to trophozoite growth. This
initial trophozoite stage measured <1mm in diam-
eter (Fig. 1E) and was observed as early as 6 h
post-inoculation, showing that host cell infection
followed by parasite development occurs rapidly
after parasite-host tissue contact.
Trophozoites on the host cell surface
48 h post-inoculation
During the invasion process, C. parvum sporozoites
always remain epicellular and appear to transform
into the trophozoite stage extracytosolic. Thus, stages
that result from host cell invasion show a similar
cellular location as gregarines, with the only differ-
ence being that Cryptosporidium becomes encapsu-
lated by a host cell apical membrane (Fig. 2A–G;
Barta and Thompson, 2006). Trophozoites shown in
Fig. 1. Oocyst excystation and sporozoite host cell invasion. (A) Intact oocyst from supernatant, 3 h post-inoculation.
(B) Oocyst excystation in vitro, 48 h post inoculation. (C) Free sporozoite isolated from supernatant 3 h
post-inoculation. (D) Free sporozoite on host cell, 7 h post-inoculation. Arrows indicate the apical regions of
sporozoites. (E) Encapsulated by the host cell apical membrane, invading sporozoites transform into the trophozoite
stage epicellularly, at 6 h and 24 h respectively. Scale bars: (A) 4 mm; (B) 2 mm; (C,D,E) 1 mm.
H. Borowski and others 16
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Fig. 2A–G may have developed from either spor-
ozoites or from merozoites type I after host cell in-
vasion. Trophozoites varied in size depending upon
their developmental stage. Early trophozoites ob-
served from 6 h post-inoculation onwards, were
f1mm in size (Fig. 1E), whereas well-developed
trophozoites observed after 24 h post-inoculation,
were up to 2.5mm (Fig. 2B). These mature tropho-
zoites appeared attached to the surface of cells, but
were separated from the host cell via an electron-
dense band (Fig. 2C). The formation of a feeder or-
ganelle structure is hypothesized for all C. parvum
stages and is shown here in Fig. 2A. Previous mor-
phological studies by Huang et al. (2004) have already
described this extracytosolic location of the parasite
and its feeder organelle attachment. Our data show
sporozoites becoming engulfed by the host cell apical
membrane. This phenomenon was proposed (Perkins
et al. 1999 ; Elliott and Clark, 2000; Pollok et al. 2003;
Chen et al. 2004a,b, 2005; Hashim et al. 2006) and
recently confirmed by Valigurova et al. (2008). It has
been suggested that the parasite induces the host cell
to encapsulate itself with the host cell apical mem-
brane (Borowski et al. 2008) to escape the hosts de-
fence mechanisms.
Trophozoites were always found to reside within
a PV and to be engulfed by the host cell apical
membrane, which displayed a smooth surface.
In later stages of trophozoite development, the basal
membrane developed a hood-like structure and cyto-
plasmic granulation occurred leading to merozoite
development within the trophozoite (Fig. 2A and B).
Single trophozoites were regularly seen attached
to infected cells (Fig. 2B), but accumulations of 2
or more trophozoites were more frequently observed
(Fig. 2D–G). The 4 trophozoites shown in Fig. 2D
are likely to have developed from sporozoites that
simultaneously excysted from a single oocyst. How-
ever, the 6 trophozoites observed in Fig. 2E are
more likely to have resulted from merozoite type I
host cell invasion, as one meront I is believed to
contain 6 or 8 merozoites (Hijjawi et al. 2001).
A merging of membranes engulfing 2 or more para-
sites was also often observed (Fig. 2D–G). This leads
to the assumption that clusters of 2 or more tropho-
zoites result from invasion of infective zoites (sporo-
zoites or merozoites) in closest proximity. Hijjawi
et al. (2004) made the observation that zoite stages
commonly accumulate in cell-free cultures. Possibly,
there is a weak unspecific binding between infective
Fig. 2. Trophozoites on the host cell surface 48 h post-inoculation. (A) Cross-section through an early trophozoite
showing the feeder-organelle (arrow) attachment to the host cell cytosol. (B) Mature trophozoite. (C) Cross-section
through a mature trophozoite revealing the electron-dense band (arrow) that separates the parasite from the
host cell. (D,E,F,G) Accumulations of trophozoites on the host cell surface. Scale bars : (A) 0.5mm; (B,C,G) 1 mm;
(D,E,F) 2 mm.
Morphological characterization of C. parvum in vitro 17
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zoites (Fig. 6A–C). Chen et al. (2000) reported that
zoite stages express surface lectins that have adhesive
properties. It is possible therefore, that these lectins
also facilitate the attachment of infective zoites to
each other. Taken together, these data suggest that
excysted sporozoites and merozoites can be adherent
to each other during host cell attachment and in-
vasion of cells (Fig. 6C). This close proximity of
invading stages results in the merging of membranes
between individuals, forming clusters of rapidly
growing trophozoites.
The effect on host cell microvilli
Protozoan parasites possess gliding motility. Gliding
motility of sporozoites has been observed in previous
studies (Barnes et al. 1998 ; Riggs et al. 1999; Wetzel
et al. 2005) leading to the suggestion that gliding
along the host cell surface is a prerequisite to host
cell invasion. C. parvum gliding trails along host cell
surfaces were observed in this study (Fig. 3A).
These gliding trails were evident as trails of elon-
gated microvilli between an excysted oocyst and
newly formed trophozoites; extending up to 15 mm.
As C. parvum is known to utilize microvilli material
to establish itself in its niche (Bonnin et al. 1995),
it can be suggested that gliding sporozoites use
microvilli material for their gliding motion and thus
cause this abnormality seen in gliding trails. As
gliding trails were not observed in all cases of oocyst
excystation, it appears that not all sporozoites glide
along the host membrane surface until they are ready
to invade a cell at a particular area. Some sporozoites
are thought to invade directly at their origin of ex-
cystation (Fig. 2F), exhibiting gliding movements
in a small area only, whereas other sporozoites might
even travel through the culture medium (Fig. 1C)
before establishing host cell contact and invading
cells some distance from excystation.
Microvillus abnormality has not only been ob-
served in gliding trails, but also around developing
trophozoites (Fig. 3B and C). Abnormally abun-
dant microvilli were frequently found to surround
trophozoites (Fig. 3B) and in some cases were
significantly elongated (Fig. 3C). As the expression
of microvilli around trophozoite clusters was found
to be higher than in non-infected areas, C. parvum
might even induce the production of microvilli to
satisfy its need for microvillar components. Micro-
villi of infected cells were also seen to appear to
Fig. 3. The parasite’s effect on host cell microvilli. (A) Gliding trail composed of elongated microvilli between
an excysted oocyst and trophozoites 3 days post-inoculation. (B,C) Abnormal microvilli clusters surround trophozoites
48 h post-inoculation. Scale bars : 2 mm.
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become incorporated into the membrane engulfing
the parasites and to aid in securing the parasite to
the host cell surface (Fig. 2B and G). This phenom-
enon has not been described before, but microvillar
material has been identified in the membrane com-
ponents engulfing the parasite using molecular tech-
niques (Bonnin et al. 1995).
Binary fission and syzygy in the C. parvum life cycle
Additional C. parvum stages were also found that
were characteristically different from those described
above. Figure 4A and B raise the possibility that
C. parvum may undergo binary fission, i.e. the
splitting of a parent cell into 2 daughter cells, other
than during the course of merogony and shizogony.
In Fig. 4A, two microgamonts that seem to be in
the process of binary fission, are engulfed by a single
membrane that may have initially encapsulated
the parent trophozoite on the host cell surface. The
finding of 2 parasites that appear to be dividing from
1 parent and seem to be the same life-cycle stage
(Fig. 4A), demonstrates that C. parvum possibly
undergoes asexual reproduction other than in the
course of merogony. As explained below, as C. par-
vum progresses through its life cycle, it appears to
become more extracellular. This is evident in stages
that appear to have little or no attachment to host
cells (Fig. 4A–C). All of these stages are likely to
be microgamonts, one of which is clearly visible in
Fig. 4A. Microgamonts occur later in the life cycle
once sexual reproduction has been initiated. Thus,
these stages show a more extracellular location. Our
observations raise the question whether C. parvum
stages with more epicellular location are still attached
to and encapsulated by the host cell apical mem-
brane, or have broken contact with host cells, but still
retain the surrounding outer host cell membrane.
Our data also indicate that C. parvum might
employ syzygy (Fig. 4C). Syzygy is defined as the
association of gamonts (pre-gametes) end-to-end or
in lateral pairing prior to the formation of gametes
that may be employed to ensure genetic diversity and
has been described in gregarines (Landers, 2001 ;
Lacombe et al. 2002; Barta and Thompson, 2006;
Toso and Omoto, 2007). Thus, it is reasonable that
syzygy may occur in closely related Cryptosporidium
species. Connecting discs between adjacent parasites
are visible (Fig. 4C and D) and these appear to be
the same as the basal discs already described in
Cryptosporidium by Valigurova et al. (2008). The
C. parvum stages involved in syzygy shown in
C
Fig. 4. Cryptosporidium parvum binary fission and syzygy. (A,B) Binary fission of C. parvum stages 4 days
post-inoculation. (C,D) C. parvum syzygy 5 days post-inoculation. Basal discs are indicated by arrows.
Scale bars : (A,B,C) 2 mm ; (D) 1 mm.
Morphological characterization of C. parvum in vitro 19
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Fig. 4C and D are probably microgamonts. As sy-
zygy was first observed when sexual life-cycle stages
occurred in culture, it can be suggested that pre-
dominantly gamont stages (here microgamonts)
employ syzygy. This correlates with findings on
gregarines and further supports the affinity of
Cryptosporidium with these apicomplexans (Landers,
2001; Toso and Omoto, 2007).
Meronts and merozoites
Trophozoites that result from sporozoite host cell
invasion develop into meronts I. Meronts I with
visible internal merozoites type I, were observed
as early as 24 h post-inoculation (Fig. 5A and B).
Like trophozoites, meronts I were found to be
attached to host cells and engulfed by the host cell
apical membrane (Fig. 5A and B). Developing mer-
onts measured approximately 1.5mm in diameter
(Fig. 5A and B), whereas mature meronts at the stage
of merozoite release were larger, measuring 2.5mm
(Fig. 5C and D). From this, it appears that tropho-
zoites developing into meronts I undergo merogony
and begin to form internal merozoites, before they
have reached their full size. Merozoites seemed to
be aligned in a parallel orientation within intact
meronts I (Fig. 5A and B) which is consistent with
observations by Hijjawi et al. (2001). Once excysted,
the merozoites type I in a meront numbered 6 or 8,
which also correlates with previous findings by
Hijjawi et al. (2001). Excysting merozoites type I
showed a rod-like shape with a pointed apical region.
In contrast to excysting oocysts, the membranes
of meronts I appear not to become perforated to
Fig. 5. Meronts I and merozoites type I. (A,B) Developing meronts I with internal merozoites type I. (C,D)
Mature meronts I at the stage of merozoite I excystation. (E,F,G) Free merozoites type I showing well-defined apical
regions (arrow) at 6 h of culture. (H) Merozoite type I host cell invasion 6 h post-inoculation. Scale bars : (A,B,C,E,F)
1mm; (D) 2 mm; (G,H) 0.5mm.
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facilitate infective zoite release. During merozoite
release, the membranes engulfing the parasite still
appeared smooth (Fig. 5C and D) and seemed to
either open up (Fig. 5 D) or rupture (Fig. 5C). When
merozoites type I are released from the meront a
residual body is left behind (Fig. 5C). Similar ob-
servations have been made on the Cryptosporidium
sp. ‘toad’ model (Valigurova et al. 2008).
Free merozoites type I were seen in cell culture
from 24 h post-inoculation onwards (Fig. 5E–H).
Merozoites, which previously were probably in-
correctly referred to as microgametes (Thompson
et al. 2005) type I were 0.4mmr1mm, with both a
rounded and a pointed end (Fig. 5E–H). The pointed
end appeared similar to that of sporozoites, which
houses the apical organelles for host cell invasion
(Tetley et al. 1998). These free merozoites must
be adhered to the host cells in some manner
(Fig. 5E–5H), otherwise they would have been
removed during sample preparation. Surface lectins
which have been detected on infective zoite stages
(Chen et al. 2000) might mediate this attachment to
host tissue. Merozoites appear initially to adhere to
host cells along their full body length, but become
stouter as cellular invasion occurs (Fig. 5H). Thus
it can be hypothesized that after initial host cell
attachment involving surface lectins, a re-orientation
of merozoite organelles occurs, bringing the apical
complex into host cell contact to initiate cellular
invasion.
Once merozoites type I are present, the parasite
employs 2 ways to replicate in its host which both
occur concurrently : (i) it progresses via asexual re-
production by the formation of meronts I and (ii) it
progresses via sexual reproduction by formation of
meronts II which then further develop into micro-
and macrogamonts (Borowski et al. 2008).
Meronts II as well as merozoites type II were
observed in culture from 72 h post-inoculation.
Meronts II were found to be bigger than meronts I
measuring 3.5mm in diameter (Fig. 6). Meronts II
appeared to possess a thicker outer membrane than
Fig. 6. Meront II and merozoites type II. (A) Excysting meront II with internal merozoites type II (arrow) 3 days
post-inoculation. (B) Merozoite type II host cell invasion. (C) Pairing of a merozoite type II with a merozoite type I 9 h
post-inoculation. Scale bars : (A) 1 mm; (B,C) 0.5mm.
Morphological characterization of C. parvum in vitro 21
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meronts I (Fig. 6A). Merozoites type II were round
and measured between 0.5mm and 1 mm in diameter
(Fig. 6A–C). Similar to meronts I, the membrane
engulfing the merozoites appeared smooth and not
perforated for their release (Fig. 6A) suggesting the
host cell origin of this membrane.
Similar to the adhesion of sporozoites to each
other via surface lectins, merozoites type I and type
II can also adhere to each other and co-invade cells
(Fig. 6B and C).
Microgamonts and macrogamonts
Merozoites type II that are released in culture,
invade cells to transform into either micro- or
macrogamonts. Four days post-inoculation micro-
and macrogamonts were observed for the first time
(Figs 7 and 8). Both gamont stages appeared to have
less contact with host cells than trophozoites or
meronts. This supports our suggestion that the fur-
ther the parasite progresses in its life cycle, the more
extracellular it appears to become. Macrogamonts
were ovular and measured about 4 mmr5mm
(Fig. 7A). Feeder organelle attachment of a macro-
gamont is visible in Fig. 7A. The surface of this
macrogamont appeared rough, suggesting that it has
already broken host cell contact leaving the host
cell-derived membrane that once surrounded it
behind. Such empty ‘ attachment-zones ’ often show
a ring-like structure (Fig. 7C), presumably where
feeding organelles were attached. These findings
correlate with observations by Valigurova et al.
(2008) who described a similar extracellular location
of macrogamonts, their detachment from host cells,
the granular structure of their feeder organelles, as
well as detachment zones in vivo, in their Crypto-
sporidium sp. ‘toad’ model.
Microgamonts were rounder and smaller than
macrogamonts, measuring about 2 mmr2mm
(Fig. 8A–C). They contained a large number of
microgametes (Fig. 8A and C), which are released to
fertilize macrogamonts. This observation is not
consistent with findings by Hijjawi et al. (2001), who
identified only 16 microgametes in 1 microgamont
with light microscopy. This difference is likely to be
due to the difficulty resolving such structures with
light microscopy. Microgametes measure about
0.1mm in diameter and are spherical. Hijjawi et al.
(2001) believed that they were non-flagellated which
is confirmed by our SEM study. The microgamonts
shown in Fig. 8A seem to be surrounded by pre-
sumably host cell-derived membrane. Fig. 8C shows
Fig. 7. Macrogamonts and their attachment zones. (A) Macrogamont with feeder organelle (arrow) 4 days
post-inoculation. (B) Developing macrogamont with feeder organelle (arrow). (C) Cryptosporidium parvum attachment
zones 5 days post-inoculation. Scale bars : (A,C) 2 mm; (B) 1 mm.
H. Borowski and others 22
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a feeder organelle attachment of the same origin as
that of the macrogamont in Fig. 7A. Approximately
half of all microgamonts seen in culture possessed
a stalk-like structure that either appeared as if the
stalk had broken (Fig. 8D) or seemed to attach the
parasite to the host cell (Fig. 8C). Again, a similar
finding has also been reported by Valigurova et al.
(2008). This stalk on microgamonts might result
from the parasite being released from the attachment
zones described above in a similar manner to macro-
gamonts. The microgamonts that possess a stalk
(Fig. 8C and D) appeared to have less host cell con-
tact compared to those which did not show such a
structure (Fig. 8A and B). Additionally, their mem-
brane appeared perforated (Fig. 8C) in contrast to
microgamonts without this stalk.
Like trophozoites, microgamonts were also ob-
served to occur in clusters of 2 or more. The ac-
cumulation of microgamonts (Fig. 8D) probably
resulted from microgamonts that had released from
their attachment zone and adhered to each other,
whereas the 2 adjacent microgamonts in Fig. 8B are
more likely to be a result of merozoite type II host
cell invasion in close proximity.
Development of C. parvum in host cell culture
without invasion of host cells
The present study is consistent with the intra-
cellular life cycle of C. parvum described by Hijjawi
et al. (2001). The stages described above all resulted
from host cell invasion, yet, complete cell-free de-
velopment of C. parvum has also been documented
at the light microscope level (Hijjawi et al. 2004)
in vitro. From our own observations, we hypothesize
that extracellular life-cycle stages might be part of
the C. parvum life cycle, or co-exist resembling
rudimentary stages of an ancestral life cycle. Our data
show that C. parvum can develop extracellularly in
the presence of host cells in an in vitro model, without
invasion. We have observed stages of C. parvum that
have been described before at the light microscopic
level but are not as yet considered as part of the life
cycle.
Life-cycle stages of C. parvum that developed
extracellularly showed a rough surface like that of
previously described free parasite stages (Fig. 5E–H
and Fig. 6A–C), and they did not appear to be
engulfed by the host cell apical membrane.
Fig. 8. Microgamonts. (A,B) Microgamonts without stalk. The arrows in A indicate a cleft along which the
macrogamonts open. (C,D) Microgamonts with stalk (arrow). Scale bars : (A,B) 1 mm; (C,D) 2 mm.
Morphological characterization of C. parvum in vitro 23
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After 48 h inoculation, we observed what we be-
lieve to be trophozoite development directly within
an oocyst (Fig. 9A). The membrane of the oocyst
releasing the trophozoites appeared rough and per-
forated, a characteristic already observed during
the release of sporozoites (Fig. 1B). Similar to these
so-called intracellular trophozoites, trophozoites
that formed directly within an oocyst measured ap-
proximately 2 mm. As these stages were observed as
early as 48 h post-inoculation (Fig. 9A) it can be
hypothesized that the trophozoites developed within
an inoculated oocyst, and were eventually released
into culture.
From our observations it can be suggested that
each sporozoite and each merozoite is capable of
undergoing merogony and transforming into a
trophozoite stage, without invasion of host cells
(Fig. 9A). This suggestion is supported by our
findings of a proposed extracellular meront 4 days
post-inoculation (Fig. 9B). The meront measured
approximately 8 mm in diameter and was round in
shape. The zoite stages observed within the meront,
measured up to 2 mm and appeared to be densely
packed. It is likely that this extracellular meront
has formed through the clumping of infective zoite
stages in culture, of which each single one has under-
gone merogony to form a trophozoite. In support of
this, accumulations of merozoites and/or tropho-
zoites, as well as the formations of meronts in cell-
free culture, have already been described (Hijjawi
et al. 2004).
Critically, how extracellular trophozoites and
meronts progress in their life cycle without cellular
invasion is still not understood. Each extracellular
trophozoite might progress in a manner typical of
an intracellular trophozoite, or each single tropho-
zoite might transform into the next life-cycle stage.
The observation that C. parvum also develops ex-
tracellularly, despite the presence of host cells, fur-
ther supports the proposal that Cryptosporidium
has a close affinity with gregarines (Carreno et al.
1999; Barta and Thompson, 2006; Valigurova et al.
2007).
CONCLUSIONS
For the first time, our study reveals the morphology
of each stage in the currently accepted life cycle of
C.parvum. The development of life-cycle stages
described here extends previous reports that were
based on light microscopic findings (Hijjawi et al.
2001, 2002, 2004). Data from our in vitro model
system are further supported by similar morpho-
logical observations from in vivo studies on C. muris
and Cryptosporidium sp. ‘ toad’ (Valigurova et al.
2008). Surprisingly, the species C. parvum observed
in our study shows more similarities to the species
Cryptosporidium sp. ‘ toad’ than to C. muris
(Valigurova et al. 2008).
Apart from revealing the morphology of accepted
life-cycle stages in C. parvum our study describes
previously unreported features of C. parvum life-
cycle stages, which include their morphological
structure and interaction with host cells. Further,
extracellular stages of C. parvum are reported and
characterized for the first time in an in vitro model
of cultured host cells at an electron microscopic
level.
It is not clear whether extracellular stages of
C. parvum occur in vivo, and whether they are part
of the parasite’s life cycle or represent rudimentary
stages of an ancestral life cycle. Future studies are
needed to identify the existence of these stages in vivo
and clarify their nature.
Fig. 9. Development of Cryptosporidium parvum in
host cell culture without the invasion of host cells.
(A) Trophozoite development within an inoculated
oocyst after 2 days. (B) Possible extracellular meront
after 4 days of culture. Scale bars : (A) 2 mm ; (B) 1 mm.
H. Borowski and others 24
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H. Borowski and others 26
... However, in contrast to reports for COLO-680N (Miller et al., 2018), no sustained parasite replication or development was observed in this host cell system. As with the well-documented parasite kinetics in HCT-8 cells [6,18,19], the number of infected cells dropped from day 2 onwards, in both cell lines, thereby reflecting incomplete sexual replication and unaccomplished karyogamy as it has been previously described [6,18]. Interestingly, a minimal but significantly higher infection rate was observed in COLO-680N cells at 4 and 5 dpi, in comparison to HCT-8, when quantifying C. parvum by VVL-based fluorescence. ...
... However, applying protocol III and thoroughly exploring kinetics, we could not find any sustained replication of C. parvum from 72 hpi onwards, neither in HCT-8 nor in COLO-680N cells. Overall, C. parvum development hardly differed in these two cell lines and observed parasite replication in both HCT-8 and COLO-680N cells, which are in agreement with previous studies performed in permanent cell lines [6,[18][19][20]24]. Moreover, significant differences in infection rates of COLO-680N and HCT-8 cell lines, by means of VVL-immunofluorescence, were possibly caused by non-specific-labelling of polypeptides left behind by parasitic stages in their intracellular but extracytoplasmatic locations. ...
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... Overall, zoonotic-relevant C. parvum is known as a microaerophilic/anaerobic parasite [22,34,35] which naturally resides in the small intestinal epithelium of various mammal species, including humans [36,37]. Of note, the human intestinal epithelium represents one of the largest body organ surfaces and constitutes 40 m 2 of interface/interaction area with the external environment and commensal and pathogenic organisms [38], rendering this unique organ into a very particular environment with its own special physiological conditions. ...
... As stated above, also C. parvum-infected host cells showed hole-like cell membrane damage after meront rupture,(Figure 2b, 16 h p.i. white arrow), evidencing that some C. parvum trophozoites underwent very fast development into mature meront stages and released merozoites in vitro. In addition, villi-like structures were also evidenced by SEM-analysis in C. parvuminfected host cells carrying meront stages(Figure 2c, 4 and 12 h p.i. black arrows) as previously reported elsewhere[37]. ...
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Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.
... Antibody labeling of C. parvum-infected HCT-8 cells has been established previously to measure C. parvum development and propagation (37). However, we aimed to characterize sporozoite invasion, which is complete in 2 h in vitro after exposure to C. parvum sporozoites (18,38,39). To differentiate sporozoite adherence and invasion, we used two identical C. parvum-specific antibodies, conjugated to different fluorophores, coupled with staining before and after cell permeabilization (see Fig. S1A in the supplemental material). ...
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Cryptosporidium infection is a leading cause of diarrhea-associated morbidity and mortality in young children, globally. Single nucleotide polymorphisms (SNP) in the human protein kinase C alpha ( PRKCA ) gene region have been associated with susceptibility to cryptosporidiosis. Here, we examined the role of protein kinase C-α (PKCα) activity in human HCT-8 intestinal epithelial cells during infection with Cryptosporidium parvum sporozoites. To delineate the role of PKCα in infection, we developed a fluorescence-based imaging assay to differentiate adherent from intracellular parasite. We tested pharmacologic agonists and antagonists of PKCα and measured the effect on C. parvum sporozoite adherence and invasion of HCT-8 cells. We demonstrated both PKCα agonists and antagonists significantly alter parasite adherence and invasion in vitro . We found HCT-8 cell PKCα is activated by C. parvum infection. Our findings suggest intestinal epithelial cell PKCα as a potential host-directed therapeutic target for cryptosporidiosis and implicate PKCα activity as a mediator of parasite adherence and invasion.
... Examination by scanning electron microscopy shows the elongation of the microvilli around the C. myocastoris developmental stages, which has also been previously observed in SCID mice infected with C. parvum [77]. Borowski et al. [78] reported the elongation of microvilli on the gliding trails of C. parvum sporozoites between an excysted oocyst and newly formed trophozoites. While the extending of microvilli in Borrowski's study was up to 15 µm, we observed much less extension. ...
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Cryptosporidium spp., common parasites of vertebrates, remain poorly studied in wildlife. This study describes the novel Cryptosporidium species adapted to nutrias (Myocastor coypus). A total of 150 faecal samples of feral nutria were collected from locations in the Czech Republic and Slovakia and examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, and gp60 loci. Molecular analyses revealed the presence of C. parvum (n = 1), C. ubiquitum subtype family XIId (n = 5) and Cryptosporidium myocastoris n. sp. XXIIa (n = 2), and XXIIb (n = 3). Only nutrias positive for C. myocastoris shed microscopically detectable oocysts, which measured 4.8–5.2 × 4.7–5.0 µm, and oocysts were infectious for experimentally infected nutrias with a prepatent period of 5–6 days, although not for mice, gerbils, or chickens. The infection was localised in jejunum and ileum without observable macroscopic changes. The microvilli adjacent to attached stages responded by elongating. Clinical signs were not observed in naturally or experimentally infected nutrias. Phylogenetic analyses at SSU, actin, and HSP70 loci demonstrated that C. myocastoris n. sp. is distinct from other valid Cryptosporidium species.
Chapter
According to the World Health Organisation, cryptosporidiosis is a global diarrhoeal disease affecting millions of individuals; it is the second most common cause of infantile death in developing countries and is increasingly identified as an emerging cause of morbidity and mortality worldwide. The disease is also extremely severe in livestock, causing profuse diarrhoea and considerable economic losses in farmed young animals. Given the lack of effective treatment (absence of vaccines and effective drugs) and the limited understanding of the causative parasite, cryptosporidiosis represents a major challenge in the battle against global diarrhoeal diseases. Currently, there are 45 described Cryptosporidium species infecting a whole spectrum of animals. In this book chapter we will present an overview of the parasite, focusing on its taxonomic status, its morphology, its prevalence and transmission. We will review both cell biological and molecular techniques currently used to investigate the biology of this parasite and we will introduce the new state-of-the-art techniques that have been established by several laboratories in the field. With the development of these new technologies, we will be able to further understand the unique biology of Cryptosporidium and its role in health and disease of its host.
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Cryptosporidium parvum (Cp) causes a gastro-intestinal disease called Cryptosporidiosis. C. parvum Inosine 5’ monophosphate dehydrogenase (CpIMPDH) is responsible for the production of guanine nucleotides. In the present study, 37 known urea-based congeneric compounds were used to build a 2D and 3D QSAR model against CpIMPDH. The built models were validated based on OECD principles. A deep learning model was adopted from a framework called Deep Purpose. The model was trained with 288 known active compounds and validated using a test set. From the training set of the 3D QSAR, a pharmacophore model was built and the best pharmacophore hypotheses were scored and sorted using a phase-hypo score. A phytochemical database was screened using both the pharmacophore model and a deep learning model. The screened compounds were considered for glide XP docking, followed by quantum polarized ligand docking. Finally, the best compound among them was considered for molecular dynamics simulation study.
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Cryptosporidium parvum is a major cause of moderate-to-severe diarrhea in humans and animals. Its compact genome contains 22 genes encoding divergent insulinase-like proteases (INS), which are poorly characterized. In this study, two small members of this family, INS-21 encoded by cgd7_2080 and INS-23 encoded by cgd5_3400, were cloned, expressed, and characterized to understand their functions. Recombinant INS-21 and INS-23 were expressed in Escherichia coli and polyclonal antibodies against these two proteins were prepared. The cgd7_2080 gene had a high transcription level during 0–2 h of in vitro C. parvum culture, while cgd5_3400 was highly transcribed at 0–6 h. INS-21 was mostly located in the apical region of sporozoites and merozoites whereas INS-23 was found as spots in sporozoites and merozoites. The immunoelectron microscopy confirmed the expression of INS-21 in the apical region of sporozoites while INS-23 appeared to be expressed in the dense granules of sporozoites. The neutralization efficiency was approximately 35%, when the cultures were treated with anti-INS23 antibodies. These results suggest that INS-21 and INS-23 are expressed in different organelles and might have different functions in the development of C. parvum .
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Introducción. Cryptosporidium parvum es un parásito zoonótico altamente prevalente, asociado a enfermedad diarreica en población inmunocomprometida, niños y terneros menores de 30 días. Esta infección puede ocasionar deshidratación, alteración del estado de conciencia, retraso en el desarrollo global y, en algunos casos, la muerte del paciente. A pesar de la alta prevalencia de C. parvum, no existen medicamentos completamente efectivos ni una vacuna aprobada para prevenir dicha enfermedad. Objetivo. Realizar una revisión de la literatura sobre candidatos vacunales contra C. parvum. Método. Revisión documental mediante la búsqueda de la literatura de los últimos 20 años, disponible en las bases de datos PubMed central, WEB OF SCIENCE, Embase, REDALYC y LILACS. Resultados. Las vacunas atenuadas, recombinantes, basadas en ADN, expresadas en vectores bacterianos y sintéticas han mostrado resultados prometedores en la inducción de inmunogenicidad contra los antígenos de C. parvum, siendo el antígeno de superficie de 15 kilodaltons de Cryptosporidium parvum (cp15), el antígeno inductor de una mejor respuesta inmune celular y humoral en el modelo murino estudiado. Conclusión. Se espera que la incorporación de nuevas técnicas para la selección de antígenos promisorios y la ejecución de una gran cantidad de ensayos in vivo, favorezcan el desarrollo de una vacuna totalmente efectiva contra C. parvum. Aunque el camino para lograr este objetivo será largo y difícil, se convierte en la mejor alternativa para controlar una de las enfermedades de interés en salud pública, con mayor impacto en la población inmunocomprometida.
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Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05) C. parvum infection when BFTE cells were pretreated for 60 min at 37 degreesC prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.
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Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05) C parvum infection when BFTE cells were pretreated for 60 min at 37 degrees C prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.
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Cryptosporidium parvum is a protozoan parasite which produces self-limited disease in immunocompetent hosts and devastating, persistent diarrhea in immunocompromised individuals. There is no effective treatment for cryptosporidiosis and little is known about the basic biology of the organism. Cloning and sequence analysis of the gene encoding GP900, a previously identified >900 kDa glycoprotein, predicts a mucin-like glycoprotein composed of distal cysteine-rich domains separated by polythreonine domains and a large membrane proximal N-glycosylated core region. A trinucleotide repeat composed predominantly of the triplet ACA encodes the threonine domains. GP900 is stored in micronemes prior to appearance on the surface of invasive forms. The concentration of native GP900 which inhibits 50% (IC50) of invasion in vitro is low picomolar; the IC50 for a recombinant cysteine rich-domain is low nanomolar. These observations indicate that GP900 is a parasite ligand for a host receptor involved in attachment/invasion and suggest that immunotherapy or chemotherapy directed against GP900 may be feasible.
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Host-cell invasion by Cryptosporidium is a complex process that requires many different factors derived from both the parasite and the host cell. However, the exact natures of the processes have yet to be resolved. Here, research on different components of the invasion process is put in context, and the sequence of events and pathways associated with the establishment of Cryptosporidium in its unique niche is clarified. In addition, initial parasite-host contact, host-cell invasion and host-cell responses are described. The roles of parasite and host-cell-derived components in the invasion process are examined, as is the question of whether Cryptosporidium actively invades cells and to what extent host-cell responses are involved.
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Two monoclonal antibodies raised against purified oocysts and excysted sporozoites of Cryptosporidium parvum identified antigens located in the anterior half of sporozoites by indirect immunofluorescence microscopic assay. The monoclonal antibodies also reacted with Triton-X-100-insoluble antigens of asexual and sexual stage parasites developing in epithelial cells in vitro and identified a 110 kilodalton antigen on immunoblots of sodium dodecyl sulfate-extracted oocysts. Immunoblotting reactivity was abolished by prior treatment of blotted antigen with periodic acid suggesting that the monoclonal antibodies recognize a carbohydrate or carbohydrate-dependent epitope(s). By immunoelectron microscopy, the antibodies reacted with a family of small, electron-dense granules located predominantly in the central region of merozoites and also with a population of cytoplasmic inclusions in macrogamonts. In addition, the monoclonal antibodies prominently labeled the parasitophorous vacuole membrane of all intracellular stages examined suggesting that the corresponding antigen(s) may be exocytosed from the granules to become associated with Triton X-100-insoluble components of the vacuolar membrane or cytoskeleton.
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Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 micrograms, 4-aminobenzoic acid 100 micrograms, calcium pantothenate 50 micrograms, ascorbic acid 875 micrograms, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously.
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Cryptosporidium parvum is a protozoan parasite which produces self-limited disease in immunocompetent hosts and devastating, persistent diarrhea in immunocompromised individuals. There is no effective treatment for cryptosporidiosis and little is known about the basic biology of the organism. Cloning and sequence analysis of the gene encoding GP900, a previously identified > 900 kDa glycoprotein, predicts a mucin-like glycoprotein composed of distal cysteine-rich domains separated by polythreonine domains and a large membrane proximal N-glycosylated core region. A trinucleotide repeat composed predominantly of the triplet ACA encodes the threonine domains. GP900 is stored in micronemes prior to appearance on the surface of invasive forms. The concentration of native GP900 which inhibits 50% (IC50) of invasion in vitro is low picomolar; the IC50 for a recombinant cysteine rich-domain is low nanomolar. These observations indicate that GP900 is a parasite ligand for a host receptor involved in attachment/invasion and suggest that immunotherapy or chemotherapy directed against GP900 may be feasible.
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Cryopreparation of live sporozoites and oocysts of the apicomplexan parasite Cryptosporidium parvum, followed by transmission electron microscopy, was undertaken to show the 3D arrangement of organelles, their number and distribution. Profiles of parasites obtained from energy-filtering transmission electron microscopy of serial sections provided 3D reconstructions from which morphometric data and stereo images were derived. The results suggest that sporozoites have a single rhoptry containing an organized lamellar body, no mitochondria or conventional Golgi apparatus, and one or two crystalline bodies. Micronemes were shown to be spherical, numerous and apically located, and to account for 0.8% of the total cell volume. Dense granules were less numerous, larger, accounted for 5.8% of the cell volume, and were located more posteriorly than micronemes. A structure juxtaposed to the nucleus with similarities to the plastid-like organelle reported for other members of the Apicomplexa was observed. The detailed analysis illustrates the advantages of cryopreparation in retaining ultrastructural fidelity of labile or difficult to preserve structures such as the sporozoite of Cryptosporidium.