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Research Focus
Cell invasion by Theileria sporozoites
Michael K. Shaw
G452 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester, UK, M13 9PT
Theileria are intracellular protozoan parasites of veterin-
ary importance, and their impact on livestock produc-
tion in many developing countries has a signicant
negative effect on the quality of human life. Host cell
invasion is a crucial aspect of Theileria biology. The
morphological events in Theileria sporozoite invasion
of bovine lymphoid cells are well established and it is
clear that the process differs signicantly from host cell
invasion in other apicomplexans, such as Plasmodium
and Toxoplasma. This article highlights some of these
differences, and compares sporozoite entry with the
entry processes of other Theileria life cycle stages.
Although details of these processes in the tick host
remain vague, it is apparent that Theileria uses different
invasion strategies in different hosts.
Theileria are tick-transmitted, intracellular apicomplexan
parasites, which infect a wide range of mammals, world-
wide [1,2]. A number of species, including Theileria parva
and Theileria annulata, are important pathogens of
domestic livestock in Old World tropical and subtropical
regions. For example, T. parva, the causative agent of East
Coast fever, costs farmers .US$ 170 million a year in
direct losses, and is a major constraint on livestock
development throughout East, central and southernAfrica
[1]. Theileria have complex life cycles that involve several
morphologically distinct developmental stages in the tick
and mammalian host cells (Fig. 1). Similar to all intra-
cellular organisms, the transmission and survival of the
parasite depends on the ability of various invasive stages
(sporozoite and merozoite in the mammalian host, the
zygote and kinete in the tick vector) to recognize and
invade specic host cells. For Theileria, most of our knowl-
edge on invasion processes comes from studies of sporo-
zoite entry by T. parva and, to a lesser extent, T. annulata.
The parasites are transmitted to mammalian hosts
when infected nymphal or adult ticks feed. The sporo-
zoites then invade lymphoid cells and induce these cells
to proliferate in an unregulated manner, leading to a
rapid clonal expansion of parasitized cells in the lymph-
oid tissues. The morphological steps in Theileria sporo-
zoite entry are illustrated in Fig. 2 (see animation at:
http://archive.bmn.com/supp/part/shaw.html). Muchof what
is known about Theileria sporozoite entry has been
reviewed in Refs [3,4]; hence, this article will focus on
some of the more unusual features of the invasion process.
Theileria sporozoite structure
The rst difference between Theileria and other
apicomplexans is the structure of the sporozoite. In
general, the invasive stages of many apicomplexan
parasites are motile, highly polarized cells (often measur-
ing 510 mminlength), with a well dened apical complex,
specialized secretory organelles (rhoptries and micro-
nemes) and an elaborate subpellicular cytoskeleton that
comprises the inner membrane complex and associated
microtubules. By contrast, Theileria sporozoites are small,
approximately spherical cells (0.751.5 mm in diameter).
Each sporozoite has a greatly reduced apical complex, and
the conoid or a similar structure, which is a characteristic
feature of some genera of Apicomplexa, is absent. The
extensive subpellicular inner membrane complex and
elaborate microtubule basket found in many motile
apicomplexan zoites is also absent in Theileria sporozoites.
Interestingly, micronemes are not present in the sporo-
zoite or merozoite, although they are found in the motile
kinete stage in the tick. In other apicomplexans, the
micronemes contain proteins that are secreted from the
apical tip of the parasite, which are important for parasite
motility and host cell invasion [5]. The absence of micro-
nemes in Theileria sporozoites and merozoites is consist-
ent with these invasive stages being non-motile.
Theileria sporozoites enter in any orientation
Theileria sporozoites are non-motile, hence the initial
sporozoitehost cell interaction is a chance event that is
not temperature-dependent. This initial contact results in
a relatively strong binding of the sporozoite to the host cell
surface and, as far as can be seen, is not reversible. The
sporozoites bind to and enter host cells in any orientation,
and invasiondoes not require re-orientation of the parasite
to bring the apical end in to close contact with the host cell
membrane. This is in contrast to other apicomplexans
which, having attached to the host cell, re-orientate in
order to enter apical-end rst. The fact that Theileria
sporozoites do not re-orientate to enter host cells apical-
end rst is probably not as a result of their lack of motility.
For example, both Plasmodiumand Toxoplasma can attach
and re-orientate to bring their apical end into contact with
the host membrane, form a tight junction and discharge
rhoptry proteins in an actin-independent way [68].
Entry by zippering
Once attached to the host surface, sporozoite entry is
accomplished by progressive circumferential zippering of
the parasite and host membranes. This zippering process
is not dependent on the actin cytoskeleton of the parasite,
does not involve the formation of a moving junction and
does not involve major re-modeling of the host cell surface
(i.e. there is no accumulation of actin laments or the Corresponding author: Michael K. Shaw (michael.k.shaw@man.ac.uk).
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formation of pseudopodia at the site of entry). Surpris-
ingly, the integrity of the host actin cytoskeleton is
essential for sporozoite binding, but sporozoite internaliz-
ation is independent of the host cytoskeleton [9]. Thus, the
zippering process is not analogous to phagocytosis by
professional phagocytes nor is it analogous to the entry of
many intracellular pathogens that requires active re-
modeling of the host actin cytoskeleton. In fact, the
physical process of zipper formation might be sufcient
in itself to cause internalization of the small spherical
sporozoite in a passive process.
Based on morphological evidence, the sporozoite surface
coat is the principal participant ininteractions withthe host
cell and it is this interaction that facilitates parasite
internalization. Becauseinotherapicomplexansthesequen-
tial discharge of the secretory organelles is essential for
successful invasion [7,1012], this raises the question as to
what is the role of the rhoptries and microspheres in
Theileria sporozoite entry. There is little evidence from
morphological studies for these secretoryorganelles playing
amajor roleintheinternalizationprocess, althoughtheyare
essential for post-entry establishment of the parasite. The
invading sporozoites do not release copious amounts of
material fromtheirrhoptriesandmicrospheres, andthehost
membrane surrounding the invading and fully internalized
parasite appears to be solely of host originand is not created
de novo from material secreted by the invading parasite.
Shedding the surface coat
One feature that appears to be common to those api-
complexan invasion processes studied to date is the
shedding of the parasite surface coat. During the zippering
process, the sporozoite surface coat (in particular, p67, a
major surface protein that is uniformly distributed on the
sporozoite surface) is shed [13]. This observation and the
evidence that protease inhibitors block entry implies that
processing of sporozoite (and/or host cell) surface mol-
ecules occurs during invasion and is vital for successful
internalization. In other apicomplexan zoites, these sur-
face proteins are sequestered in the micronemes and
rhoptries at rst, and then relocated to the surface during
gliding and after parasite binding. During secretion, some
of these surface proteins undergo either autoproteolytic
cleavage or are processed by proteases sequestered in the
secretory organelles. Proteolytic processing is, thus, essen-
tial for secretion of functional surface molecules, in addi-
tion to their subsequent shedding. However, there is no
evidence for secretion of surface proteins in non-motile
Theileria sporozoites, or for a role of the rhoptries or
microspheres in the internalization process. Thus, the
Fig. 1. The Theileria life cycle. Theileria sporozoites are inoculated into a mammalian host when an infected tick takes a meal. The sporozoites invade lymphoid cells, and
each parasite develops into a multinucleate syncytial schizont (a). At the same time, the parasite induces host cell transformation and proliferation (b). A proportion of schi-
zonts differentiates into merozoites and these invade erythrocytes (c). For some species, there are multiple rounds of asexual division in the lymphocyte stage, with little or
no multiplication in the erythrocytes (e.g. Theileria parva), whereas in other species, there is little or no intralymphocytic multiplication, and the parasite multiplies almost
exclusively in the erythrocytes (e.g. Theileria mutans) (d). Ticks become infected by ingesting infected erythrocytes, and gametogenesis and fertilization takes place in the
gut lumen (e). The resulting zygote invades a gut epithelial cell where it remains during the tick moult cycle and develops into a single motile kinete (f). The motile kinete
egresses the gut cell and invades the salivary glands (g). Tick feeding initiates rapid sporozoite development in the salivary glands, and infective sporozoites are emitted
during the later stages of feeding (h). Transmission in the tick is trans-stadial in that larvae or nymphs can become infected. The parasites survive in the gut epithelium
during the moulting cycle, and are transmitted to another mammalian host when the resulting post-moult nymphs or adults feed. For more details, see Refs [1,2]. Repro-
duced, with permission, from Ref. [4].
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
In the tick host In the mammalian host
Salivary glands
Gut epithelial cell
Gut lumen
Schizonts in
lymphoid cells
Piroplasms in erythrocytes
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identity and source of the protease(s) involved in the
shedding of p67 remains unknown.
Post-entry development
At 37 8C, the process of sporozoite binding and internal-
ization occurs within ,3 mins. This is much slower than
Toxoplasma tachyzoites, which actively enter cells in
1520 secs and, presumably reects the mainly passive
nature of the zippering process. All of the newly internal-
ized sporozoites are completely surrounded by the closely
apposed host cell membrane. However, unlike many api-
complexans, the Theileria sporozoite escapes from the
enclosing host membrane to lie freely in the host cyto-
plasm. Failure to escape within 1530 mins following
entry results in the death of the parasite, suggesting that
the parasite does not have the ability to modify the
enclosing host membrane (c.f. Toxoplasma and Plasmo-
dium) [8,14,15]. It is noteworthy that all of the Theileria
intracellular stages (the schizont in the lymphoblast, the
intra-erythrocytic piroplasms, the zygote kinete in the tick
gut cells, and the sporoblast in the tick salivary gland cell)
are free in the host cytoplasm and do not develop inside a
parasitophorous vacuole. However, there is no evidence that
Theileria sporozoites and merozoites can actively invade
hostcellsbythephysical disruptionof thehost membrane, as
has been described for some other apicomplexans [16]. It is,
however, possible that one or more Theileria stages in the
tick host could actively enter and/or exit host cells by
physically disrupting the host membrane [4].
The escape into the host cytoplasm is preceded by the
separation of the enclosing host membrane from the
sporozoite surface. This process and the subsequent dis-
solution of the host membrane coincides with the dis-
charge of the rhoptries and microspheres, and the
appearance of a thick layer (1015 nm) of fuzzy material
on the surface of the parasite. In addition, as the parasite
escapes into the host cytoplasm, a regular array of host
cell-derived microtubules becomes associated with material
secreted from the microspheres onto the parasite surface.
Thus, the secretory organelles in Theileria sporozoites
participate primarily in the escape and establishment of
the parasite within the host cytoplasm, rather than in the
entry process. This is distinct from the roles played by the
secretory organelles in other apicomplexans where they
Fig. 2. The entry and establishment of Theileria parva sporozoites into bovine lymphocytes. This process involves a dened series of events, as follows: (a) the initial recog-
nition and binding of the sporozoite to the host cell surface, which leads to the formation (b) of a close, continual junction between the sporozoite and host cell membranes.
A ,6 nm thick layer of denser material separates these two membranes. The progressive circumferential zippering of the two closely apposed sporozoite and host cell
membranes, concomitant with the loss of the sporozoite surface coat and the movement of the parasite into the host cell, results in the parasite becoming fully internalized
within the host cell. The parasite is still surrounded by the closely apposed host surface membrane (c). The entry process is pH-dependent, requires the participation of live,
intact parasites and host cells, but does not require de novo protein synthesis. (d) The tightly apposed host and parasite membranes become separated at the same time as the
discharge of the rhoptries and microspheres, and the appearance of a 1015 nm thick layer of denser material on the parasite surface. The dissolution of the host membrane
releases the parasite into the host cell cytoplasm. This is followed by the formation of an orderly array of host cell-derived microtubules around the parasite that is associated
with the microsphere-derived material on the surface of the parasite (e,f). The process of escape occurs at a neutral pH and is not dependent on the acidication of the parasite-
containing vacuole. For an animated version of this life cycle, go to: http://archive.bmn.com/supp/part/shaw.html. Reproduced, with permission, from Ref. [30].
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
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participate, in a sequential manner, in parasite gliding, in
the entry process, and in the formation and modication of
the parasitophorous vacuole membrane [7,1012].
At present, little is known about the molecular com-
ponents of Theileria secretory organelles compared with
other apicomplexans [11]. Furthermore, the mechanism(s)
controlling discharge of the rhoptries and microspheres of
Theileria sporozoites is not known. The available evidence
implies that zippering initiates some sort of clock mechan-
ism, which leads to the discharge of the organelles. How-
ever, discharge is not induced by Ca
2
and is not controlled
by a Ca
2
-dependent pathway [17], unlike, for example,
microneme secretion in Toxoplasma [18].
By escaping into the host cytoplasm, the parasite will
avoid many of the problems associated with living in a
parasitophorous vacuole [19,20]. Whereas many intra-
cellular pathogens interact with and/or remodel the host
cell actincytoskeleton[21,22], theassociationof theschizont
with the host microtubule system is more unusual. The
association of the parasite with the host cell microtubules
probably facilitates the movement of the parasite to the
perinuclear region of the host cell [23]. It is clear that some
viruses can recruit host cell motor molecules to their sur-
face and use the host microtubule network for intracellular
transport [24]. However, as the schizont develops and
throughout its intracellular life, the orderly array of host
microtubules remains associated with the schizont surface.
Infact, thesemicrotubules are highlystable: treatment with
microtubule-disruptingagents, suchas nocodazole, does not
affect these parasite-associated microtubules, but it does
disrupt other host cell microtubule systems. Only at the
onset of merogony do the microtubules disappear from the
surface of the schizont, implying that the parasite is
responsible for maintaining the association. However, the
nature of this interaction(s) and what parasite proteins
interact with the microtubules is not known. Studies aimed
at understandingthisunusual parasitehost cell association
could help us understand more about microtubule biology.
Theileria merozoites entering bovine erythrocytes
Once established in the host cytoplasm, the parasite
differentiates into a multinucleate schizont and, at the
same time, induces host cell transformation leading to
clonal expansion of infected cells. To continue its life cycle,
the schizont undergoes a differentiation and cellulariza-
tion process to produce uninucleate merozoites [25], which
are then liberated into the bloodstream where they invade
erythrocytes. Merozoite invasionoccurs ina similar manner
to sporozoite entry, withthe parasite subsequently escaping
from the enclosing erythrocyte membrane to be free in the
erythrocyte cytoplasm [26]. The similarities in sporozoite
and merozoite entry indicate that common molecular mech-
anismsmight beinvolvedintheprocesses, but littleisknown
about the natureandtimingof these molecular interactions.
Theileria invading tick cells
Host cell invasion by Theileria sporozoites and merozoites
represents one of the simplest forms of entry described
for apicomplexan parasites. Whether this represents the
original method of host cell invasion by apicomplexans or
is a secondary reduction is not known. In the tick, the
parasite invades and develops in gut epithelial cells and in
the salivary gland (Fig 1). Whereas neither invasion
process has been described, it is known that the kinete,
which develops in the tick gut epithelial cell, is motile and
moves by a smooth gliding motion similar to that seen in
other apicomplexans. Structurally, the kinete resembles
motile zoites of other apicomplexans and is distinct from
the sporozoite and merozoite stages. Thus, Theileria clearly
possesses the genetic information necessary to differentiate
into a motile invasive stage, which makes the question of
why the sporozoite and merozoite stages employ such a
simplied method of entry even more intriguing. Interest-
ingly, recent studies on Plasmodium have highlighted that
themethodsof hostcell invasionemployedbyapicomplexans
are much more diverse than initially thought [16,27].
Perspective
A major constraint to studying Theileria invasion pro-
cesses is the availability of suitable material, and only a
few laboratories have the facilities or resources to main-
tain the Theileria life cycle. Currently, work on the sporo-
zoite invasion process focuses on characterizing parasite
surface molecules that could prove suitable for use in
diagnostic tests and/or in vaccine development. However,
there are several novel features of the schizont stage that
are of wider biological interest, such as the interaction of
the parasite with the host microtubule system. A second
unusual feature of Theileria biology is that the intracellu-
lar parasite induces and maintains host cell transform-
ation, which leads to clonal expansion of infected cells.
Whereas the molecular mechanisms underlying host cell
transformation are becoming clearer [28], how transform-
ation is initiated is not understood. Do signalling processes
triggered during entry lead directly to transformation? Is
transformation a separate event initiated only after the
parasite is established in the host cytoplasm? Interest-
ingly, the proportion of susceptible lymphocytes can be
increased experimentally by incubation with compounds
that induce lymphocyte proliferation [29]. One possibility is
that lymphocytes activated in vivo are the target cells, and
the parasite subsequently takes advantage of the intra-
cellular signaling pathways that were already activated.
Sporozoites can also invade and develop into schizonts
in other non-lymphoid cells present at the tick attachment
site, although host cell transformation does not occur. This
suggests that sporozoite entry and early development and
host cell transformation could represent two separate pro-
cesses. A corollary to this is that, for Theileria, host cell
specicity operates at a number of levels within the
parasitehost association.
Hopefully, these unusual features and the imminent
availability of the Theileria genome sequence (http://www.
tigr.org/tdb/e2k1/tpa1/; http://www.sanger.ac.uk/Projects/
T_annulata/) will encourage more cell biologists to become
interested in this intriguing and fascinating parasite.
Acknowledgements
M.S. acknowledges Alan S. Bowman (University of
Aberdeen, UK) for his help with the animation.
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Trends in teaching parasitology:
the American situation
Alex D.W. Acholonu
Department of Biological Sciences, Alcorn State University, Alcorn State, MS 39096, USA
Parasitic infections in both humans and animals are still
rampant and appear to be increasing. There is a need
for parasitologists, human and animal doctors to con-
tribute toward the global eradication of communicable
and food-borne diseases. The need for teaching parasit-
ology, the recommendations and future perspectives
are discussed in this article, and it is proposed that
macrobiology should be recognized and taught as a
subject area to include the study of eukaryotic organ-
isms encompassing macroparasites.
Parasitology deals with the study of relationships between
parasites and their biotic and abiotic environments,
between parasites and their hosts, and their reactions
towards each other. Parasitismis a way of life in which one
species gains its livelihood at the expense of another [13].
Parasitic organisms range from 10 meter-long tapeworms
to the enigmatic prions that appear to comprise protein,
with a bewildering array inbetween, fromprokaryotes and
protists to metazoans which occupy every ecological niche
imaginable [2]. In short, parasitology includes the study
of microorganisms (studied as microbiology) and macro-
organisms (which, logically, could and should be studied
as macrobiology, as long as microbiology is used and
1471-4922/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(02)00002-8
Corresponding author: Alex D.W. Acholonu (acholonu@lorman.alcorn.edu).
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