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The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife? Neil Portman and Jan Sˇlapeta Faculty of Veterinary Science, University of Sydney, Sydney, Australia
Apicomplexa are an ancient group of single-celled pathogens of humans and animals that include the etiological agents of such devastating plagues as malaria, toxoplasmosis, and coccidiosis. The defining feature of the Apicomplexa is the apical complex, the invasion machinery used to gain access to host cells. Evidence gathered from apicomplexans and their closest relatives argues that the apical complex is an extreme example of flagellum adaptability. The value of non-apicomplexan models, such as Chromera velia, is considered in an effort to understand the modern apical complex. The origin of the apical complex is unknown, but recent evidence points to a remarkable contribution from the flagellum to its evolution. Apicomplexans: bringing together two iconic structures Apicomplexan pathogens (see Glossary) are so successful that there is no vertebrate or invertebrate which is not parasitised by at least one species in this group. They form the most diverse group of single-celled pathogens and are responsible for numerous medically and commercially important diseases, including malaria, toxoplasmosis, and coccidiosis (Figure 1). The group is defined by the possession of an apical complex, a collection of cytoskeletal elements and secretory systems used to gain access to host cells. The apical complex is not present in hosts parasitised by Apicomplexa, and is therefore a potential target for drug and vaccine therapeutic intervention. On the other end of the conservation scale is the eukaryotic flagellum, present in the last common ancestor of all eukaryotes, and which is still found across all major eukaryotic phyla today. The Apicomplexa might be considered something of a backwater for flagellar function; many lineages do not build a flagellum and, for those that do, it is restricted to male gametes [1] (Box 1). However, recent work has started to elucidate a remarkable confluence of these two iconic structures [2,3], and it now seems that the flagellum plays Corresponding authors: Portman, N. ([email protected]); Sˇlapeta, J. ([email protected]). Keywords: Apicomplexa; Chromera; apical complex; conoid; flagellum; rootlet. 1471-4922/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2013.12.006
a much more important part in the success of apicomplexan pathogens than previously thought. A tool for all occasions The solution to many problems encountered during eukaryotic evolution has been the flagellum. Flagella are employed in a vast diversity of conserved and specific roles across eukaryotic phyla [4–9]. In general concept, the flagellum might be considered to be the eukaryotic Swiss Army knife. The tools of such an implement would then be the specific combinations of flagellar form and function exhibited between different lineages, and even between cells of the same organism. Flagellate cells can have anything from one flagellum, as found in trypanosomes and mammalian primary cilia, to the two flagella of Chlamydomonas reinhardtii, to the hundreds of flagella per cell possessed by the ciliates Paramecium spp. and Tetrahymena spp. and by the ciliated airway epithelium in mammals. At the core of the canonical flagellum is a microtubular axoneme in which nine doublet microtubules surround a central pair of singlet microtubules, known as the 9+2 arrangement. Axonemes are templated from the flagellar basal body, one of the pivotal microtubule-organising centres. In many organisms, basal bodies also play a role during mitosis, where they are known as centrioles. In some phyla, the exact same structure cycles between roles in the flagellum and then in mitosis during daughter-cell formation [10–12]. The 9+2 arrangement of axonemal microtubules can be found across diverse eukaryotic organisms and is usually associated with a function in motility, as in the quintessential model flagellate C. reinhardtii, the kinetoplastid parasite Trypanosoma brucei, or male gametes in many eukaryotes. However, motility and 9+2 are only the starting points for the functionality and diversity of the flagellum. Flagella have well-established roles in signal transduction and environmental sensing. This can be in addition to, or as an intrinsic part of, a role in motility – as in the case of the mechanosensing and mating behaviour in C. reinhardtii [13,14] – but in many instances flagella have been specifically adapted to a sensory and signalling role at the expense of active motility. Some famous examples of this are the well-studied immotile sensory cilia of neuronal cells of Caenorhabditis elegans [15]. These are referred to as having a 9+0 arrangement because they have nine outer doublets but lack the central pair of microtubules. The Trends in Parasitology xx (2013) 1–7
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Glossary
flagellar structure is modified to such an extent that it is hardly recognisable, in this case to accommodate the multilayered membrane discs of the phototransduction machinery [17].
Apical complex: a defining feature of Apicomplexa that comprises a system of structural and secretory elements. Facilitates interaction with the host cell. The main structures in the complex include the rhoptries, micronemes, apical polar ring, and conoid. Apical polar ring (APR): pivotal component of apical complex of all Apicomplexa; a microtubule-organizing centre. Nucleates the subpellicular microtubules. Apicomplexa: a phylum of single-cell parasites of medical and veterinary importance; comprises three main parasite groups: haematozoans, coccidians, and gregarines, that evolved from a free-living photosynthetic ancestor. Axoneme: extends from the flagellar basal body; typically consists of nine doublet microtubules organised longitudinally into a circle surrounding a central pair of singlet microtubules (9+2). The main structural and functional component of the flagellum. Basal body (centriole): nucleates the flagellum of eukaryotes; a microtubuleorganizing centre. In metazoans and other lineages it also has a role in mitosis as part of the centrosome; typically consists of 9 triplet microtubules ordered in a circle (9+0). Coccidia: a group of intracellular parasites of vertebrates and invertebrates whose apical complex possess a conoid; includes Toxoplasma gondii and Eimeria spp. Conoid: small cone-shaped structure of the apical complex composed of a spiral of unique proto-filaments with a mechanical role in invasion of host cells in coccidians and gregarines. Flagellum: a defining feature of eukaryotes that is composed of basal body and axoneme; in addition to locomotion, the flagellum has many other conserved and specific functions across eukaryotic phyla. Gregarines: a group of diverse extracellular parasites of invertebrates that possess conoids. Haematozoa: a group of intracellular parasites of vertebrates whose apical complex lacks a conoid; parasites transmitted by vectors, including Plasmodium spp. causing malaria and Babesia spp. causing tick fever. Intraconoidal microtubules: single pair of microtubules occurring in the centre of the apical polar ring and conoid. Myzocytosis: a feeding strategy employed by colpodellids and some early branching Apicomplexa in which the cell uses its apical complex to suck the cytoplasm from its prey. Paraflagellar rod: the extra-axonemal structure of kinetoplastids. The paraflagellar rod lies alongside the axoneme and functions in flagellar motility. Perkinsids: parasites of bivalves possessing flagella and features resembling the apical complex. Phototransduction machinery: specialised membrane domains of sensory cilia in retinal rod and cone cells used for detection of light. Protofilaments: polymers of a- and b-tubulin heterodimers arranged end-toend to form filaments. Thirteen protofilaments interacting along the long axes form the canonical microtubule. The arrangement of the tubulin dimers gives the protofilaments polarity, and this is conferred to the microtubule because all protofilaments are orientated in the same direction. Rhoptries and micronemes: secretory organelles containing proteins required for parasite motility, adhesion, and invasion of host cells, and for the establishment of the parasites within the host cell. Rootlet: complex of bundles of microtubules originating from around the basal-body region of the flagellum. SAS6-like (SAS6L): a novel protein with significant homology to the basalbody protein spindle assembly defective 6 (SAS-6). SAS6L has been localised to the apical complex of T. gondii and to the basal-body apparatus of T. brucei. Striated fibre assemblins: proteins that form fibres that organise flagellar basal bodies and anchor them to the rest of the cytoskeleton, in other words to microtubules. Subpellicular microtubules: microtubules radiating from the apical polar ring under the cell surface that are responsible for the elongated structure and cell polarity of Apicomplexa.
primary cilia of mammalian cells are also of the immotile 9+0 type. These important cilia have a central role in coordinating the key developmental signal transduction pathways hedgehog and Wnt [5,6]. Flagella are also notable for the diversity of elaborations and deviations from the ancestral 9+2 state. These can range from the loss of certain elements, including the previously mentioned 9+0 axonemes, to the incorporation of large extraaxonemal structures, with the paraflagellar rod of trypanosomatids being an elegant and well-known example of this [16]. In some highly specialised cases, such as the sensory cilia of mammalian photoreceptor cells, the 2
The parasite’s toolkit Where the flagellum is conserved across eukaryotic phyla, the apical complex is the defining feature of the apicomplexans, and under some definitions is considered to be restricted to this group. The apical complex is a system of structural and secretory elements that evolved from the feeding apparatus of the free-living ancestors of the apicomplexans (Figure 2) [1,18]. The main role of this system is in host–parasite interactions including host-cell invasion. This role in host-cell invasion means that the apical complex is present in all stages of the life cycle except the male gametes, a marked counterpoint to the presence of the flagellum through the life cycle, such that the two structures are never present in the same cell at the same time (Box 1). The foundation of the apical complex is the apical polar ring (APR), one of the key microtubule-organising centres in Apicomplexa that resides at the apical tip of invasive cells [1]. Emanating from the APR is a set of subpellicular microtubules that vary in number between species and even between life-cycle stages within species, although not between cells of the same species during the same life-cycle stage. These microtubules extend posteriorly into the cell beneath the plasma membrane in a spiral configuration, conferring shape and structure to the cell body. Other key conserved elements of the apical complex are the rhoptries and micronemes, secretory organelles that deliver the enzymes necessary for the establishment and maintenance of host-cell infections [19]. In the coccidian and gregarine lineages the APR is also associated with a conoid [1]. This remarkable structure is a solid coneshaped association of tightly spiralled fibres generated by an unusual polymer of tubulin. Whereas the conoid fibres are composed of tubulin, the protofilaments adopt a novel, open comma-shaped configuration very different from the closed circular aspect of microtubules [20]. Although the conoid is absent from several key lineages, including Plasmodium spp. and the piroplasms, its presence in early branching apicomplexans, such as the gregarines [21], suggests that these are secondary losses and that the ancestral apicomplexan possessed a conoidal apical complex. A flagellar contribution to the apical complex Apicomplexans evolved from free-living, photosynthetic (probably bi-)flagellate algae, as evidenced by their retention of a relict plastid, the apicoplast (Figure 1) [22]. It has long been thought that apicomplexans had mostly dispensed with their flagella. However, mounting evidence suggests that far from losing the flagellum in the apical complex, and particularly in the manner of formation and apportioning during daughter-cell biogenesis, we may be observing one of the most diverged examples of flagellum adaptability [2,3]. In many organisms, flagellar basal bodies are associated with rootlets, sets of microtubules and other elements, such as striated fibres, that nucleate near the basal body
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Haematozoa Babesia spp. Theileria spp.
Plasmodoium spp. Leucocytozoon spp.
Coccidia
Piroplasms
Hemosporidia malarial parasites
200
Dixenic (invertebrates and vertebrates)
Hepatozoon spp. Hemogregarines (invertebrates)
300
Eimeria spp. Monoxenic coccidia (vertebrates)
Cryptosporidia Cryptosporidium spp.
500 Host cell
Monoxenic (vertebrates)
2500
Gregarina spp.
1700
50
Maesia spp.
Gregarines
Arch
igre
?
gar
ines
ms
diu leni
Se
6000
Obligate parasites
Monoxenic (invertebrates)
Colpodellids
Pr Agam ot oc ococ oc cid cid ia ia
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pp.
Apicomplexa Complete apical complex (conoid, rhoptry, micronemes) Prey
Colpodella spp.
Toxoplasma gondii Cyst formning coccidia (vertebrates)
?
Eugregarines
Neogregarines
250
30
2
10
Chromerids Chromera velia Vitrella brassicaformis Photoautotrophs associated with corals
Marine and freshwater predators
Key: Loss of conoid Loss of plasd
Photosynthec ancestor (bi-flagellate and free-living) TRENDS in Parasitology
Figure 1. Apicomplexa and their closest sisters: colpodellids and chromerids. Schematic representations depicting the relationship and diversity (thickness of branches) of the dominant groups based on formally identified species (approximate number on branches). Question marks indicate uncertainty in branching order. Apicomplexan, colpodellid, and chromerid outlines document the presence or absence of flagella and the feeding strategy of the group. Host organisms for Apicomplexa are indicated in brackets.
Box 1. The flagellum and the Apicomplexa Despite its seemingly endless adaptability and utility as a platform for cellular processes, and their evolutionary origins as flagellate algae, the Apicomplexa seem to have eschewed the benefits of the flagellum for the most part. Some lineages, including the piroplasms (Theileria spp., Babesia spp.) and Cryptosporidium spp., have dispensed with a flagellum altogether, whereas in all other lineages it is entirely restricted to a brief existence in male gametes where it is primarily used for motility. Most organisms that build a flagellum use the same conserved mechanism, intraflagellar transport (IFT) [36], to construct it and to maintain it once built. IFT involves the transport of flagellar components to and from the tip of the growing flagellum (anterograde and retrograde transport, respectively), which extends outward from the cell body within its own specialised membrane. This process is sometimes referred to as compartmentalised flagellar biogenesis. Flagellar biogenesis in the Apicomplexa is somewhat different. Some lineages, such as T. gondii, do employ IFT and build compartmentalised flagella, although in this case one of the crucial motors involved in retrograde transport is absent [37]. This perhaps reflects the relatively short lifespan of flagella in these organisms and a reduced need for flagellar maintenance. Other lineages, for instance the haematozoa and gregarines, employ an unusual method of assembling axonemes in the cytoplasm [38]. Plasmodium spp., which produce uniflagellate microgametes, have dispensed with IFT entirely [39]. Orthologues to all known genes encoding IFT-related proteins are absent from the genome. Axonemes are constructed in the cytoplasm in a quiescent state and then activated as the last step in gametocytogenesis [38]. The mechanisms involved in constructing and patterning axonemes in this way, and the process of flagellar activation, so far remain as important unanswered questions.
and play crucial roles in organising and positioning subcellular structures. In the African trypanosome, T. brucei, for example, the flagellar rootlet consists of four specialised microtubules that integrate into the subpellicular array of microtubules and play a crucial role in passing morphological patterning information from the mother cell to the two daughter cells [23]. In C. reinhardtii the rootlet microtubules are associated with a striated fibre consisting of striated fibre assemblin (SFA) that organises and orientates the basal bodies both in interphase, where they subtend flagella, and during mitosis, where they are part of the centrosome [10]. Despite their general lack of flagella, apicomplexans encode several SFA-related proteins [24], and recent work is leading to an understanding of the surprising role they play in daughter-cell budding in T. gondii [3]. Apicomplexans employ an unusual mode of cell division that can yield two (e.g., Toxoplasma spp.) to hundreds (e.g., Eimeria spp.) of daughter cells from a single mother [25]. In the mother cell multiple rounds of mitosis can occur before the resulting genomes are each matched with a new apical complex and a full set of organelles, and are packaged into daughter buds. Recently, some of the mechanisms behind the fidelity of this matching process have been brought to light [3]. These investigators demonstrated that, during daughter-cell formation, a fibre composed of SFA proteins arises near a pair of centrioles in a similar way to which the homologous structure in C. reinhardtii is associated with 3
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Chromera velia
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(C)
(B)
(D)
Key:
Conoid fibre
Subpellicular mt
Flagellum
Plasma membrane
Intraconoidal mt
Rootlet
Rhoptry
Polar rings TRENDS in Parasitology
Figure 2. Cartoon representation of the apical complex of Toxoplasma gondii and Chromera velia. Transverse views of the apex of the apical complex (A,C) and longitudinal projections (B,D). In T. gondii (A,B) the conoid (brown) is a tightly coiled, closed structure comprised unusual polymers of tubulin. It is topped by a pair of polar rings (dark blue) and subtended by the apical polar ring (dark blue), one of the key microtubule-organising centres in the cell. Within the lumen of the conoid are a pair of intraconoidal microtubules (green) and the apical ends of the rhoptries (grey), secretory organelles that deliver the enzymes necessary for the parasite to establish itself in the host cell. The microtubules that form the subpellicular cytoskeleton (orange) extend from the apical polar ring towards the posterior of the cell. In C. velia (C,D) the conoid (brown) comprises microtubules and is open along the side proximal to the flagellar groove. The microtubular rootlet (pink) that originates at the adjacent basal body (light blue) partially covers the open side of the conoid at its apex. As with T. gondii, the conoid surrounds a pair of intraconoidal microtubules (green) and a so-far uncharacterised membrane-bound system extends into the conoidal lumen (grey). Abbreviation: mt, microtubule.
the flagellar basal bodies. During mitosis, the centrioles of T. gondii duplicate with the nucleus such that each nascent nucleus is associated with a pair of centrioles [26]. These centrioles are positioned in close proximity to the spindleorganising plaques that nucleate the mitotic spindle microtubules. The SFA fibre extends from between the centrioles, and is closely associated with the nascent apical complex of the new daughter cell, looping around the extreme apical end of the conoid. Not only does the SFA fibre tether a post-mitotic nucleus to a nascent apical complex, but the lack of daughter-cell apical complex formation in cells depleted of SFA fibres suggests that it also has a crucial role in templating the new apical complex at the very beginning of the budding process. In this way, a mechanism used by the algal ancestors of apicomplexans to organise basal bodies and flagella has been repurposed in T. gondii to match organelles and nuclei to daughter buds. The potential for this mechanism to scale with the number of daughter cells to be produced, and hence address the question of matching daughter buds to organelles across apicomplexans, is compelling. The centrioles are associated with the nucleus via the spindle-organising plaque, and at each mitosis are also duplicated. With the 4
deployment of the SFA fibre, each post-mitotic nucleus can template and tether their own apical organelles. So far this system has only been demonstrated in T. gondii, although a fibre with similar positioning and cell-cycle regulation has been noted in Plasmodium [27]. Recently, SAS6-like (SAS6L) was localised to the apical tip of the conoid in T. gondii and to the basal body of T. brucei [2]. A second localisation site was also identified in T. brucei that may correlate with the position of the proximal portion of the flagellar rootlet in this organism. These authors postulate two evolutionary possibilities to explain the data: (i) a complete subsumption of the flagellum into the conoid such that the SAS6L localisation observed in T. gondii is derived from the basal body, or (ii) an incorporation of elements of the flagellar rootlet into the conoid. A third possibility is that the SAS6L protein has been coopted into the conoid in Toxoplasma, without any contribution from other basal body-associated elements, perhaps through a more general tubulin-interacting capacity. Localising SAS6L in a non-apicomplexan that produces both a conoid and a flagellum contemporaneously may answer the question of its origins in the T. gondii conoid, and possibly speak to the origins of the conoid itself.
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Box 2. Sister groups of Apicomplexa Chromerids: Chromera velia is an autotrophic protist that was isolated from Australian stony corals in Sydney Harbour and on One Tree Island, Great Barrier Reef (Figure I) [30]. C. velia can be easily cultured and studied in the laboratory, and have an apical complex consisting of an open conoid and a putative endomembrane organelle [32]. Two or four daughter cells bud from a mother cell. C. velia possesses a functional chloroplast, which is homologous to the vestigial non-photosynthetic plastid of Apicomplexa. Phylogenetic analyses confirm that C. velia is closely related to Apicomplexa [30,40].
Colpodellids: Colpodella pugnax is a predatory protist present in abundance in artificial hypersaline lagoons in South Australia (Figure II) [41]. C. pugnax is maintained in the laboratory by the addition of the green alga, Dunaliella spp., as food. C. pugnax possesses an apical complex that includes an open-sided conoid, used in myzocytosis of prey, and rhoptry-like structures [29,42]. Two or four daughter cells bud from a mother cell, and morphological studies have found no evidence of a vestigial plastid. Phylogenetic analysis confirms the sister relationship of Colpodella spp. to parasitic Apicomplexa [34,43].
TRENDS in Parasitology TRENDS in Parasitology
Figure I. One Tree Island, Great Barrier Reef, Australia. Chromera velia was isolated from the stony coral of the Great Barrier Reef in 2001. Image courtesy of One Tree Island Research Station, The University of Sydney.
Origins of the apical complex–flagellum association Repurposing of the flagellar apparatus and the specialisation of the conoid in T. gondii are faits accomplis. What then are the opportunities to investigate the origins of these structures? Although the apical complex is considered to be the defining feature of the Apicomplexa, several related lineages, for instance the perkinsids and some early-diverging dinoflagellates, also exhibit elements of these structures [28]. We can get even closer to the possible ancestral state of the apical complex found in the Apicomplexa by examining their closest relatives, the colpodellids and Chromera velia (Figure 1, Box 2). These two groups diverged from Apicomplexa, and from each other, at around the same time as obligate parasitism emerged in Apicomplexa. Colpodellids are free-living predatory protists that deploy their apical complex in a feeding behaviour known as myzocytosis, which is also employed by some Apicomplexa [29]. C. velia is a free-living marine autotroph that is thought to be a potential symbiont of corals [30]. Very little is known about the possible function of the apical complex of C. velia: it may play a similar role to the apicomplexan apical complex in facilitating the entry of the cell into coral hosts, or it may be used as an endocytic aid to feeding as with the colpodellids. Of course, it may also have some completely novel function peculiar to C. velia because we also know very little about the life cycle of this organism in its natural habitat. In culture, C. velia exists predominantly as aflagellate coccoid cysts [30–32]. Two, four, or more bi-flagellate daughter cells can be produced as a response to stimulus by light as part of a dial cycle [30,32]. In the production of four or
Figure II. Hypersaline artificial lagoons in South Australia. Colpodella pugnax and the green alga Dunaliella spp. are abundant in these artificial lagoons. Image reproduced courtesy of Google (Map data 2014 Google).
more daughter cells, as with Apicomplexa, the parental genome undergoes multiple rounds of mitosis before the initiation of daughter-cell budding [32]. At the earliest stages of daughter bud formation, basal bodies are present at the periphery of the cell and have nucleated axonemes. As a stark counterpoint to the situation in Apicomplexa, it is only the flagellate forms in these organisms that contain an apical complex [32,33]. Here we will focus on C. velia, although for the most part the pertinent features are very similar between the two groups. The apical complex of C. velia centres around an array of what appear to be canonical microtubules, variously termed the conoid, the open conoid, or the pseudo-conoid (Figure 2C,D) [31,32]. In addition to apparently being composed of microtubules, this conoid differs from that seen in Apicomplexa in that it is open along one side. Similarly to the apicomplexan conoid, it is associated with a pair of intraconoidal microtubules and a large membrane-bound system that occupies a position comparable to that of the rhoptries. Recent evidence shows that the conoid is positioned in the pointed anterior end of flagellate cells and is always adjacent to the shorter of the two flagella of C. velia [32,33]. The basal body of this flagellum is associated with a microtubular rootlet that lies alongside the conoid. A similar association of conoid and rootlet has been reported in colpodellids and other alveolates [28,34,35]. In a transverse view at the apical end of the conoid, the rootlet microtubules fill the gap in the opensided cone, albeit set slightly outside the conoidal circumference. 5
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Opinion Rootlet microtubules close the conoid: path to the perfect pathogen We hypothesise that, in the ancestral apicomplexan, these rootlet microtubules became integrated with the microtubules of the adjacent open conoid, in effect closing the conoid and beginning evolution of the structure we see in Apicomplexa today. This model might account for the apparent association of the T. gondii SFA fibre with both the conoid and the centriole. So far, there are no reports of striated fibres associated with the flagellar rootlet in C. velia, but given the phylogenetic distribution of SFA homologues it is likely that this protein will be expressed by this organism. By comparison with C. reinhardtii, we predict that, if this is the case, C. velia SFA will colocalise with the rootlet microtubules. We therefore envisage an evolutionary process whereby the microtubules of the flagellar rootlet integrate into the conoid, closing it and setting it on the path to the modern structure. This includes the conversion of the conoidal microtubules into the unusual form observed in T. gondii, most likely through the addition of hitherto unidentified microtubule associated proteins [20]. The former rootlet microtubules retain an attachment to an SFA fibre that connects them to the flagellar basal body, perhaps by the action of a SAS6L orthologue [2]. As the ancestral apicomplexan adopts an obligate parasitic lifestyle, the need for the flagellum is lost, and the basal bodies fail to develop in most life-cycle stages. The stalled basal bodies remain in the cytoplasm rather than migrating to the cell periphery during daughter-cell formation, but retain their connection to the former rootlet microtubules, now an integral part of the conoid, via the SFA fibre during daughter-cell biogenesis [3]. In proposing this model we recognise that there remain important unanswered questions regarding the homology of the apical complex of Apicomplexa to those found in other alveolates. Evidence of such homology currently comprises ultrastructural similarities of position and gross morphology of components, and some indications of homologous function – as in the case of the myzocytosis employed by colpodellids and apicomplexan gregarines. To test our proposed model, the hypothesis that these structures are homologous, and are not the result of convergence, further molecular data and a greater understanding of daughter-cell formation in C. velia and colpodellids at the ultrastructural level will be required. The expression and behaviour of some of the proteins discussed here – SAS6L, SFA, and other well-known markers of apical structures in Apicomplexa – will be crucial data if we are to determine the extent to which these free-living sister groups offer a snap-shot of early apicomplexan evolution in terms of the apical complex. Concluding remarks It is becoming more apparent that in the apical complex Apicomplexa may have added yet another tool to the eukaryotic Swiss Army knife. The flagellum has been studied extensively in numerous organisms over many years, and there is now the potential to leverage this wealth of knowledge to identify and understand novel 6
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mechanisms and components of apical complex structure, function, and inheritance. These recent findings also highlight the need for model organisms that give insight into the evolutionary history of the apicomplexans. C. velia is easily cultured and has a genome project underway. However, there is an urgent need for the development of molecular toolkits and transfection systems to exploit fully the potential of these organisms. The full extent of the flagellar contribution to the apical complex, and whether the SFA-based matching mechanism is widespread among apicomplexans or an oddity of T. gondii, remain important unanswered questions. Acknowledgements Support for N.P. is provided by the University of Sydney Postdoctoral Research Fellowship scheme.
References 1 Morrissette, N.S. and Sibley, L.D. (2002) Cytoskeleton of apicomplexan parasites. Microbiol. Mol. Biol. Rev. 66, 21–38 2 Cruz de Leon, J. et al. (2013) A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components. Eukaryot. Cell 12, 1009–1019 3 Francia, M.E. et al. (2012) Cell division in Apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella. PLoS Biol. 10, e1001444 4 Mitchell, D.R. (2007) The evolution of eukaryotic cilia and flagella as motile and sensory organelles. Adv. Exp. Med. Biol. 607, 130–140 5 Christensen, S.T. et al. (2007) Sensory cilia and integration of signal transduction in human health and disease. Traffic 8, 97–109 6 Goetz, S.C. and Anderson, K.V. (2010) The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344 7 Vaughan, S. (2010) Assembly of the flagellum and its role in cell morphogenesis in Trypanosoma brucei. Curr. Opin. Microbiol. 13, 453–458 8 Satir, P. and Christensen, S.T. (2007) Overview of structure and function of mammalian cilia. Annu. Rev. Physiol. 69, 377–400 9 Ginger, M.L. et al. (2008) Swimming with protists: perception, motility and flagellum assembly. Nat. Rev. Microbiol. 6, 838–850 10 Dutcher, S.K. (2003) Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii. Traffic 4, 443–451 11 Christensen, S.T. et al. (2008) The primary cilium coordinates signaling pathways in cell cycle control and migration during development and tissue repair. Curr. Top. Dev. Biol. 85, 261–301 12 Dawe, H.R. et al. (2007) Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells. J. Cell Sci. 120, 7–15 13 Fujiu, K. et al. (2011) Mechanoreception in motile flagella of Chlamydomonas. Nat. Cell Biol. 13, 630–632 14 Pan, J. et al. (2003) Protein transport and signal transduction during fertilization in Chlamydomonas. Traffic 4, 452–459 15 Inglis, P.N. et al. (2007) The sensory cilia of Caenorhabditis elegans. WormBook 1–22 16 Portman, N. and Gull, K. (2010) The paraflagellar rod of kinetoplastid parasites: from structure to components and function. Int. J. Parasitol. 40, 135–148 17 Fliegauf, M. et al. (2007) When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880–893 18 Hu, K. et al. (2006) Cytoskeletal components of an invasion machine – the apical complex of Toxoplasma gondii. PLoS Pathog. 2, e13 19 Bradley, P.J. and Sibley, L.D. (2007) Rhoptries: an arsenal of secreted virulence factors. Curr. Opin. Microbiol. 10, 582–587 20 Hu, K. et al. (2002) A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J. Cell Biol. 156, 1039–1050 21 Leander, B.S. (2008) Marine gregarines: evolutionary prelude to the apicomplexan radiation? Trends Parasitol. 24, 60–67 22 McFadden, G.I. (2011) The apicoplast. Protoplasma 248, 641–650 23 Vaughan, S. and Gull, K. (2008) The structural mechanics of cell division in Trypanosoma brucei. Biochem. Soc. Trans. 36, 421–424 24 Lechtreck, K.F. (2003) Striated fiber assemblin in apicomplexan parasites. Mol. Biochem. Parasitol. 128, 95–99
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Opinion 25 Gubbels, M.J. et al. (2008) The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched? Int. J. Parasitol. 38, 1343– 1358 26 Anderson-White, B. et al. (2012) Cytoskeleton assembly in Toxoplasma gondii cell division. Int. Rev. Cell Mol. Biol. 298, 1–31 27 Schre´vel, J. et al. (2008) Vesicle trafficking during sporozoite development in Plasmodium berghei: ultrastructural evidence for a novel trafficking mechanism. Parasitology 135, 1–12 28 Okamoto, N. et al. (2012) Description of two species of early branching dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp. PLoS ONE 7, e34900 29 Leander, B.S. and Keeling, P.J. (2003) Morphostasis in alveolate evolution. Trends Ecol. Evol. 18, 395–402 30 Moore, R.B. et al. (2008) A photosynthetic alveolate closely related to apicomplexan parasites. Nature 451, 959–963 31 Obornı´k, M. et al. (2011) Morphology and ultrastructure of multiple life cycle stages of the photosynthetic relative of apicomplexa, Chromera velia. Protist 162, 115–130 32 Portman, N. et al. (2014) Evidence of intraflagellar transport and apical complex formation in a free-living relative of the Apicomplexa. Eukaryot. Cell 13, 10–20 33 Weatherby, K. et al. (2011) Surface and flagella morphology of the motile form of Chromera velia revealed by field-emission scanning electron microscopy. Protist 162, 142–153 34 Leander, B.S. et al. (2003) Molecular phylogeny and surface morphology of Colpodella edax (Alveolata): insights into the
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phagotrophic ancestry of apicomplexans. J. Eukaryot. Microbiol. 50, 334–340 Brugerolle, G. (2002) Colpodella vorax: ultrastructure, predation, lifecycle mitosis, and phylogenetic relationships. Eur. J. Protistol. 38, 113– 125 Kozminski, K.G. et al. (1993) A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc. Natl. Acad. Sci. U. S. A. 90, 5519– 5523 Wickstead, B. and Gull, K. (2007) Dyneins across eukaryotes: a comparative genomic analysis. Traffic 8, 1708–1721 Sinden, R.E. et al. (2010) The flagellum in malarial parasites. Curr. Opin. Microbiol. 13, 491–500 Briggs, L.J. et al. (2004) More than one way to build a flagellum: comparative genomics of parasitic protozoa. Curr. Biol. 14, R611–612 Obornı´k, M. et al. (2009) Evolution of the apicoplast and its hosts: From heterotrophy to autotrophy and back again. Int. J. Parasitol. 39, 1–12 Simpson, A.G.B. and Patterson, D.J. (1996) Ultrastructure and identification of the predatory flagellate Colpodella pugnax Cienkowski (Apicomplexa) with a description of Colpodella turpis n. sp. and a review of the genus. Syst. Parasitol. 33, 187–198 Cavalier-Smith, T. and Chao, E.E. (2004) Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.). Eur. J. Protistol. 40, 185–212 Kuvardina, O.N. et al. (2002) The phylogeny of colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the freeliving sister group to apicomplexans. J. Euk. Microbiol. 49, 498–504
7
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The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife? Neil Portman and Jan Sˇlapeta Faculty of Veterinary Science, University of Sydney, Sydney, Australia
Apicomplexa are an ancient group of single-celled pathogens of humans and animals that include the etiological agents of such devastating plagues as malaria, toxoplasmosis, and coccidiosis. The defining feature of the Apicomplexa is the apical complex, the invasion machinery used to gain access to host cells. Evidence gathered from apicomplexans and their closest relatives argues that the apical complex is an extreme example of flagellum adaptability. The value of non-apicomplexan models, such as Chromera velia, is considered in an effort to understand the modern apical complex. The origin of the apical complex is unknown, but recent evidence points to a remarkable contribution from the flagellum to its evolution. Apicomplexans: bringing together two iconic structures Apicomplexan pathogens (see Glossary) are so successful that there is no vertebrate or invertebrate which is not parasitised by at least one species in this group. They form the most diverse group of single-celled pathogens and are responsible for numerous medically and commercially important diseases, including malaria, toxoplasmosis, and coccidiosis (Figure 1). The group is defined by the possession of an apical complex, a collection of cytoskeletal elements and secretory systems used to gain access to host cells. The apical complex is not present in hosts parasitised by Apicomplexa, and is therefore a potential target for drug and vaccine therapeutic intervention. On the other end of the conservation scale is the eukaryotic flagellum, present in the last common ancestor of all eukaryotes, and which is still found across all major eukaryotic phyla today. The Apicomplexa might be considered something of a backwater for flagellar function; many lineages do not build a flagellum and, for those that do, it is restricted to male gametes [1] (Box 1). However, recent work has started to elucidate a remarkable confluence of these two iconic structures [2,3], and it now seems that the flagellum plays Corresponding authors: Portman, N. ([email protected]); Sˇlapeta, J. ([email protected]). Keywords: Apicomplexa; Chromera; apical complex; conoid; flagellum; rootlet. 1471-4922/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2013.12.006
a much more important part in the success of apicomplexan pathogens than previously thought. A tool for all occasions The solution to many problems encountered during eukaryotic evolution has been the flagellum. Flagella are employed in a vast diversity of conserved and specific roles across eukaryotic phyla [4–9]. In general concept, the flagellum might be considered to be the eukaryotic Swiss Army knife. The tools of such an implement would then be the specific combinations of flagellar form and function exhibited between different lineages, and even between cells of the same organism. Flagellate cells can have anything from one flagellum, as found in trypanosomes and mammalian primary cilia, to the two flagella of Chlamydomonas reinhardtii, to the hundreds of flagella per cell possessed by the ciliates Paramecium spp. and Tetrahymena spp. and by the ciliated airway epithelium in mammals. At the core of the canonical flagellum is a microtubular axoneme in which nine doublet microtubules surround a central pair of singlet microtubules, known as the 9+2 arrangement. Axonemes are templated from the flagellar basal body, one of the pivotal microtubule-organising centres. In many organisms, basal bodies also play a role during mitosis, where they are known as centrioles. In some phyla, the exact same structure cycles between roles in the flagellum and then in mitosis during daughter-cell formation [10–12]. The 9+2 arrangement of axonemal microtubules can be found across diverse eukaryotic organisms and is usually associated with a function in motility, as in the quintessential model flagellate C. reinhardtii, the kinetoplastid parasite Trypanosoma brucei, or male gametes in many eukaryotes. However, motility and 9+2 are only the starting points for the functionality and diversity of the flagellum. Flagella have well-established roles in signal transduction and environmental sensing. This can be in addition to, or as an intrinsic part of, a role in motility – as in the case of the mechanosensing and mating behaviour in C. reinhardtii [13,14] – but in many instances flagella have been specifically adapted to a sensory and signalling role at the expense of active motility. Some famous examples of this are the well-studied immotile sensory cilia of neuronal cells of Caenorhabditis elegans [15]. These are referred to as having a 9+0 arrangement because they have nine outer doublets but lack the central pair of microtubules. The Trends in Parasitology xx (2013) 1–7
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Glossary
flagellar structure is modified to such an extent that it is hardly recognisable, in this case to accommodate the multilayered membrane discs of the phototransduction machinery [17].
Apical complex: a defining feature of Apicomplexa that comprises a system of structural and secretory elements. Facilitates interaction with the host cell. The main structures in the complex include the rhoptries, micronemes, apical polar ring, and conoid. Apical polar ring (APR): pivotal component of apical complex of all Apicomplexa; a microtubule-organizing centre. Nucleates the subpellicular microtubules. Apicomplexa: a phylum of single-cell parasites of medical and veterinary importance; comprises three main parasite groups: haematozoans, coccidians, and gregarines, that evolved from a free-living photosynthetic ancestor. Axoneme: extends from the flagellar basal body; typically consists of nine doublet microtubules organised longitudinally into a circle surrounding a central pair of singlet microtubules (9+2). The main structural and functional component of the flagellum. Basal body (centriole): nucleates the flagellum of eukaryotes; a microtubuleorganizing centre. In metazoans and other lineages it also has a role in mitosis as part of the centrosome; typically consists of 9 triplet microtubules ordered in a circle (9+0). Coccidia: a group of intracellular parasites of vertebrates and invertebrates whose apical complex possess a conoid; includes Toxoplasma gondii and Eimeria spp. Conoid: small cone-shaped structure of the apical complex composed of a spiral of unique proto-filaments with a mechanical role in invasion of host cells in coccidians and gregarines. Flagellum: a defining feature of eukaryotes that is composed of basal body and axoneme; in addition to locomotion, the flagellum has many other conserved and specific functions across eukaryotic phyla. Gregarines: a group of diverse extracellular parasites of invertebrates that possess conoids. Haematozoa: a group of intracellular parasites of vertebrates whose apical complex lacks a conoid; parasites transmitted by vectors, including Plasmodium spp. causing malaria and Babesia spp. causing tick fever. Intraconoidal microtubules: single pair of microtubules occurring in the centre of the apical polar ring and conoid. Myzocytosis: a feeding strategy employed by colpodellids and some early branching Apicomplexa in which the cell uses its apical complex to suck the cytoplasm from its prey. Paraflagellar rod: the extra-axonemal structure of kinetoplastids. The paraflagellar rod lies alongside the axoneme and functions in flagellar motility. Perkinsids: parasites of bivalves possessing flagella and features resembling the apical complex. Phototransduction machinery: specialised membrane domains of sensory cilia in retinal rod and cone cells used for detection of light. Protofilaments: polymers of a- and b-tubulin heterodimers arranged end-toend to form filaments. Thirteen protofilaments interacting along the long axes form the canonical microtubule. The arrangement of the tubulin dimers gives the protofilaments polarity, and this is conferred to the microtubule because all protofilaments are orientated in the same direction. Rhoptries and micronemes: secretory organelles containing proteins required for parasite motility, adhesion, and invasion of host cells, and for the establishment of the parasites within the host cell. Rootlet: complex of bundles of microtubules originating from around the basal-body region of the flagellum. SAS6-like (SAS6L): a novel protein with significant homology to the basalbody protein spindle assembly defective 6 (SAS-6). SAS6L has been localised to the apical complex of T. gondii and to the basal-body apparatus of T. brucei. Striated fibre assemblins: proteins that form fibres that organise flagellar basal bodies and anchor them to the rest of the cytoskeleton, in other words to microtubules. Subpellicular microtubules: microtubules radiating from the apical polar ring under the cell surface that are responsible for the elongated structure and cell polarity of Apicomplexa.
primary cilia of mammalian cells are also of the immotile 9+0 type. These important cilia have a central role in coordinating the key developmental signal transduction pathways hedgehog and Wnt [5,6]. Flagella are also notable for the diversity of elaborations and deviations from the ancestral 9+2 state. These can range from the loss of certain elements, including the previously mentioned 9+0 axonemes, to the incorporation of large extraaxonemal structures, with the paraflagellar rod of trypanosomatids being an elegant and well-known example of this [16]. In some highly specialised cases, such as the sensory cilia of mammalian photoreceptor cells, the 2
The parasite’s toolkit Where the flagellum is conserved across eukaryotic phyla, the apical complex is the defining feature of the apicomplexans, and under some definitions is considered to be restricted to this group. The apical complex is a system of structural and secretory elements that evolved from the feeding apparatus of the free-living ancestors of the apicomplexans (Figure 2) [1,18]. The main role of this system is in host–parasite interactions including host-cell invasion. This role in host-cell invasion means that the apical complex is present in all stages of the life cycle except the male gametes, a marked counterpoint to the presence of the flagellum through the life cycle, such that the two structures are never present in the same cell at the same time (Box 1). The foundation of the apical complex is the apical polar ring (APR), one of the key microtubule-organising centres in Apicomplexa that resides at the apical tip of invasive cells [1]. Emanating from the APR is a set of subpellicular microtubules that vary in number between species and even between life-cycle stages within species, although not between cells of the same species during the same life-cycle stage. These microtubules extend posteriorly into the cell beneath the plasma membrane in a spiral configuration, conferring shape and structure to the cell body. Other key conserved elements of the apical complex are the rhoptries and micronemes, secretory organelles that deliver the enzymes necessary for the establishment and maintenance of host-cell infections [19]. In the coccidian and gregarine lineages the APR is also associated with a conoid [1]. This remarkable structure is a solid coneshaped association of tightly spiralled fibres generated by an unusual polymer of tubulin. Whereas the conoid fibres are composed of tubulin, the protofilaments adopt a novel, open comma-shaped configuration very different from the closed circular aspect of microtubules [20]. Although the conoid is absent from several key lineages, including Plasmodium spp. and the piroplasms, its presence in early branching apicomplexans, such as the gregarines [21], suggests that these are secondary losses and that the ancestral apicomplexan possessed a conoidal apical complex. A flagellar contribution to the apical complex Apicomplexans evolved from free-living, photosynthetic (probably bi-)flagellate algae, as evidenced by their retention of a relict plastid, the apicoplast (Figure 1) [22]. It has long been thought that apicomplexans had mostly dispensed with their flagella. However, mounting evidence suggests that far from losing the flagellum in the apical complex, and particularly in the manner of formation and apportioning during daughter-cell biogenesis, we may be observing one of the most diverged examples of flagellum adaptability [2,3]. In many organisms, flagellar basal bodies are associated with rootlets, sets of microtubules and other elements, such as striated fibres, that nucleate near the basal body
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Haematozoa Babesia spp. Theileria spp.
Plasmodoium spp. Leucocytozoon spp.
Coccidia
Piroplasms
Hemosporidia malarial parasites
200
Dixenic (invertebrates and vertebrates)
Hepatozoon spp. Hemogregarines (invertebrates)
300
Eimeria spp. Monoxenic coccidia (vertebrates)
Cryptosporidia Cryptosporidium spp.
500 Host cell
Monoxenic (vertebrates)
2500
Gregarina spp.
1700
50
Maesia spp.
Gregarines
Arch
igre
?
gar
ines
ms
diu leni
Se
6000
Obligate parasites
Monoxenic (invertebrates)
Colpodellids
Pr Agam ot oc ococ oc cid cid ia ia
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Apicomplexa Complete apical complex (conoid, rhoptry, micronemes) Prey
Colpodella spp.
Toxoplasma gondii Cyst formning coccidia (vertebrates)
?
Eugregarines
Neogregarines
250
30
2
10
Chromerids Chromera velia Vitrella brassicaformis Photoautotrophs associated with corals
Marine and freshwater predators
Key: Loss of conoid Loss of plasd
Photosynthec ancestor (bi-flagellate and free-living) TRENDS in Parasitology
Figure 1. Apicomplexa and their closest sisters: colpodellids and chromerids. Schematic representations depicting the relationship and diversity (thickness of branches) of the dominant groups based on formally identified species (approximate number on branches). Question marks indicate uncertainty in branching order. Apicomplexan, colpodellid, and chromerid outlines document the presence or absence of flagella and the feeding strategy of the group. Host organisms for Apicomplexa are indicated in brackets.
Box 1. The flagellum and the Apicomplexa Despite its seemingly endless adaptability and utility as a platform for cellular processes, and their evolutionary origins as flagellate algae, the Apicomplexa seem to have eschewed the benefits of the flagellum for the most part. Some lineages, including the piroplasms (Theileria spp., Babesia spp.) and Cryptosporidium spp., have dispensed with a flagellum altogether, whereas in all other lineages it is entirely restricted to a brief existence in male gametes where it is primarily used for motility. Most organisms that build a flagellum use the same conserved mechanism, intraflagellar transport (IFT) [36], to construct it and to maintain it once built. IFT involves the transport of flagellar components to and from the tip of the growing flagellum (anterograde and retrograde transport, respectively), which extends outward from the cell body within its own specialised membrane. This process is sometimes referred to as compartmentalised flagellar biogenesis. Flagellar biogenesis in the Apicomplexa is somewhat different. Some lineages, such as T. gondii, do employ IFT and build compartmentalised flagella, although in this case one of the crucial motors involved in retrograde transport is absent [37]. This perhaps reflects the relatively short lifespan of flagella in these organisms and a reduced need for flagellar maintenance. Other lineages, for instance the haematozoa and gregarines, employ an unusual method of assembling axonemes in the cytoplasm [38]. Plasmodium spp., which produce uniflagellate microgametes, have dispensed with IFT entirely [39]. Orthologues to all known genes encoding IFT-related proteins are absent from the genome. Axonemes are constructed in the cytoplasm in a quiescent state and then activated as the last step in gametocytogenesis [38]. The mechanisms involved in constructing and patterning axonemes in this way, and the process of flagellar activation, so far remain as important unanswered questions.
and play crucial roles in organising and positioning subcellular structures. In the African trypanosome, T. brucei, for example, the flagellar rootlet consists of four specialised microtubules that integrate into the subpellicular array of microtubules and play a crucial role in passing morphological patterning information from the mother cell to the two daughter cells [23]. In C. reinhardtii the rootlet microtubules are associated with a striated fibre consisting of striated fibre assemblin (SFA) that organises and orientates the basal bodies both in interphase, where they subtend flagella, and during mitosis, where they are part of the centrosome [10]. Despite their general lack of flagella, apicomplexans encode several SFA-related proteins [24], and recent work is leading to an understanding of the surprising role they play in daughter-cell budding in T. gondii [3]. Apicomplexans employ an unusual mode of cell division that can yield two (e.g., Toxoplasma spp.) to hundreds (e.g., Eimeria spp.) of daughter cells from a single mother [25]. In the mother cell multiple rounds of mitosis can occur before the resulting genomes are each matched with a new apical complex and a full set of organelles, and are packaged into daughter buds. Recently, some of the mechanisms behind the fidelity of this matching process have been brought to light [3]. These investigators demonstrated that, during daughter-cell formation, a fibre composed of SFA proteins arises near a pair of centrioles in a similar way to which the homologous structure in C. reinhardtii is associated with 3
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Toxoplasma gondii
Chromera velia
(A)
(C)
(B)
(D)
Key:
Conoid fibre
Subpellicular mt
Flagellum
Plasma membrane
Intraconoidal mt
Rootlet
Rhoptry
Polar rings TRENDS in Parasitology
Figure 2. Cartoon representation of the apical complex of Toxoplasma gondii and Chromera velia. Transverse views of the apex of the apical complex (A,C) and longitudinal projections (B,D). In T. gondii (A,B) the conoid (brown) is a tightly coiled, closed structure comprised unusual polymers of tubulin. It is topped by a pair of polar rings (dark blue) and subtended by the apical polar ring (dark blue), one of the key microtubule-organising centres in the cell. Within the lumen of the conoid are a pair of intraconoidal microtubules (green) and the apical ends of the rhoptries (grey), secretory organelles that deliver the enzymes necessary for the parasite to establish itself in the host cell. The microtubules that form the subpellicular cytoskeleton (orange) extend from the apical polar ring towards the posterior of the cell. In C. velia (C,D) the conoid (brown) comprises microtubules and is open along the side proximal to the flagellar groove. The microtubular rootlet (pink) that originates at the adjacent basal body (light blue) partially covers the open side of the conoid at its apex. As with T. gondii, the conoid surrounds a pair of intraconoidal microtubules (green) and a so-far uncharacterised membrane-bound system extends into the conoidal lumen (grey). Abbreviation: mt, microtubule.
the flagellar basal bodies. During mitosis, the centrioles of T. gondii duplicate with the nucleus such that each nascent nucleus is associated with a pair of centrioles [26]. These centrioles are positioned in close proximity to the spindleorganising plaques that nucleate the mitotic spindle microtubules. The SFA fibre extends from between the centrioles, and is closely associated with the nascent apical complex of the new daughter cell, looping around the extreme apical end of the conoid. Not only does the SFA fibre tether a post-mitotic nucleus to a nascent apical complex, but the lack of daughter-cell apical complex formation in cells depleted of SFA fibres suggests that it also has a crucial role in templating the new apical complex at the very beginning of the budding process. In this way, a mechanism used by the algal ancestors of apicomplexans to organise basal bodies and flagella has been repurposed in T. gondii to match organelles and nuclei to daughter buds. The potential for this mechanism to scale with the number of daughter cells to be produced, and hence address the question of matching daughter buds to organelles across apicomplexans, is compelling. The centrioles are associated with the nucleus via the spindle-organising plaque, and at each mitosis are also duplicated. With the 4
deployment of the SFA fibre, each post-mitotic nucleus can template and tether their own apical organelles. So far this system has only been demonstrated in T. gondii, although a fibre with similar positioning and cell-cycle regulation has been noted in Plasmodium [27]. Recently, SAS6-like (SAS6L) was localised to the apical tip of the conoid in T. gondii and to the basal body of T. brucei [2]. A second localisation site was also identified in T. brucei that may correlate with the position of the proximal portion of the flagellar rootlet in this organism. These authors postulate two evolutionary possibilities to explain the data: (i) a complete subsumption of the flagellum into the conoid such that the SAS6L localisation observed in T. gondii is derived from the basal body, or (ii) an incorporation of elements of the flagellar rootlet into the conoid. A third possibility is that the SAS6L protein has been coopted into the conoid in Toxoplasma, without any contribution from other basal body-associated elements, perhaps through a more general tubulin-interacting capacity. Localising SAS6L in a non-apicomplexan that produces both a conoid and a flagellum contemporaneously may answer the question of its origins in the T. gondii conoid, and possibly speak to the origins of the conoid itself.
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Box 2. Sister groups of Apicomplexa Chromerids: Chromera velia is an autotrophic protist that was isolated from Australian stony corals in Sydney Harbour and on One Tree Island, Great Barrier Reef (Figure I) [30]. C. velia can be easily cultured and studied in the laboratory, and have an apical complex consisting of an open conoid and a putative endomembrane organelle [32]. Two or four daughter cells bud from a mother cell. C. velia possesses a functional chloroplast, which is homologous to the vestigial non-photosynthetic plastid of Apicomplexa. Phylogenetic analyses confirm that C. velia is closely related to Apicomplexa [30,40].
Colpodellids: Colpodella pugnax is a predatory protist present in abundance in artificial hypersaline lagoons in South Australia (Figure II) [41]. C. pugnax is maintained in the laboratory by the addition of the green alga, Dunaliella spp., as food. C. pugnax possesses an apical complex that includes an open-sided conoid, used in myzocytosis of prey, and rhoptry-like structures [29,42]. Two or four daughter cells bud from a mother cell, and morphological studies have found no evidence of a vestigial plastid. Phylogenetic analysis confirms the sister relationship of Colpodella spp. to parasitic Apicomplexa [34,43].
TRENDS in Parasitology TRENDS in Parasitology
Figure I. One Tree Island, Great Barrier Reef, Australia. Chromera velia was isolated from the stony coral of the Great Barrier Reef in 2001. Image courtesy of One Tree Island Research Station, The University of Sydney.
Origins of the apical complex–flagellum association Repurposing of the flagellar apparatus and the specialisation of the conoid in T. gondii are faits accomplis. What then are the opportunities to investigate the origins of these structures? Although the apical complex is considered to be the defining feature of the Apicomplexa, several related lineages, for instance the perkinsids and some early-diverging dinoflagellates, also exhibit elements of these structures [28]. We can get even closer to the possible ancestral state of the apical complex found in the Apicomplexa by examining their closest relatives, the colpodellids and Chromera velia (Figure 1, Box 2). These two groups diverged from Apicomplexa, and from each other, at around the same time as obligate parasitism emerged in Apicomplexa. Colpodellids are free-living predatory protists that deploy their apical complex in a feeding behaviour known as myzocytosis, which is also employed by some Apicomplexa [29]. C. velia is a free-living marine autotroph that is thought to be a potential symbiont of corals [30]. Very little is known about the possible function of the apical complex of C. velia: it may play a similar role to the apicomplexan apical complex in facilitating the entry of the cell into coral hosts, or it may be used as an endocytic aid to feeding as with the colpodellids. Of course, it may also have some completely novel function peculiar to C. velia because we also know very little about the life cycle of this organism in its natural habitat. In culture, C. velia exists predominantly as aflagellate coccoid cysts [30–32]. Two, four, or more bi-flagellate daughter cells can be produced as a response to stimulus by light as part of a dial cycle [30,32]. In the production of four or
Figure II. Hypersaline artificial lagoons in South Australia. Colpodella pugnax and the green alga Dunaliella spp. are abundant in these artificial lagoons. Image reproduced courtesy of Google (Map data 2014 Google).
more daughter cells, as with Apicomplexa, the parental genome undergoes multiple rounds of mitosis before the initiation of daughter-cell budding [32]. At the earliest stages of daughter bud formation, basal bodies are present at the periphery of the cell and have nucleated axonemes. As a stark counterpoint to the situation in Apicomplexa, it is only the flagellate forms in these organisms that contain an apical complex [32,33]. Here we will focus on C. velia, although for the most part the pertinent features are very similar between the two groups. The apical complex of C. velia centres around an array of what appear to be canonical microtubules, variously termed the conoid, the open conoid, or the pseudo-conoid (Figure 2C,D) [31,32]. In addition to apparently being composed of microtubules, this conoid differs from that seen in Apicomplexa in that it is open along one side. Similarly to the apicomplexan conoid, it is associated with a pair of intraconoidal microtubules and a large membrane-bound system that occupies a position comparable to that of the rhoptries. Recent evidence shows that the conoid is positioned in the pointed anterior end of flagellate cells and is always adjacent to the shorter of the two flagella of C. velia [32,33]. The basal body of this flagellum is associated with a microtubular rootlet that lies alongside the conoid. A similar association of conoid and rootlet has been reported in colpodellids and other alveolates [28,34,35]. In a transverse view at the apical end of the conoid, the rootlet microtubules fill the gap in the opensided cone, albeit set slightly outside the conoidal circumference. 5
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Opinion Rootlet microtubules close the conoid: path to the perfect pathogen We hypothesise that, in the ancestral apicomplexan, these rootlet microtubules became integrated with the microtubules of the adjacent open conoid, in effect closing the conoid and beginning evolution of the structure we see in Apicomplexa today. This model might account for the apparent association of the T. gondii SFA fibre with both the conoid and the centriole. So far, there are no reports of striated fibres associated with the flagellar rootlet in C. velia, but given the phylogenetic distribution of SFA homologues it is likely that this protein will be expressed by this organism. By comparison with C. reinhardtii, we predict that, if this is the case, C. velia SFA will colocalise with the rootlet microtubules. We therefore envisage an evolutionary process whereby the microtubules of the flagellar rootlet integrate into the conoid, closing it and setting it on the path to the modern structure. This includes the conversion of the conoidal microtubules into the unusual form observed in T. gondii, most likely through the addition of hitherto unidentified microtubule associated proteins [20]. The former rootlet microtubules retain an attachment to an SFA fibre that connects them to the flagellar basal body, perhaps by the action of a SAS6L orthologue [2]. As the ancestral apicomplexan adopts an obligate parasitic lifestyle, the need for the flagellum is lost, and the basal bodies fail to develop in most life-cycle stages. The stalled basal bodies remain in the cytoplasm rather than migrating to the cell periphery during daughter-cell formation, but retain their connection to the former rootlet microtubules, now an integral part of the conoid, via the SFA fibre during daughter-cell biogenesis [3]. In proposing this model we recognise that there remain important unanswered questions regarding the homology of the apical complex of Apicomplexa to those found in other alveolates. Evidence of such homology currently comprises ultrastructural similarities of position and gross morphology of components, and some indications of homologous function – as in the case of the myzocytosis employed by colpodellids and apicomplexan gregarines. To test our proposed model, the hypothesis that these structures are homologous, and are not the result of convergence, further molecular data and a greater understanding of daughter-cell formation in C. velia and colpodellids at the ultrastructural level will be required. The expression and behaviour of some of the proteins discussed here – SAS6L, SFA, and other well-known markers of apical structures in Apicomplexa – will be crucial data if we are to determine the extent to which these free-living sister groups offer a snap-shot of early apicomplexan evolution in terms of the apical complex. Concluding remarks It is becoming more apparent that in the apical complex Apicomplexa may have added yet another tool to the eukaryotic Swiss Army knife. The flagellum has been studied extensively in numerous organisms over many years, and there is now the potential to leverage this wealth of knowledge to identify and understand novel 6
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mechanisms and components of apical complex structure, function, and inheritance. These recent findings also highlight the need for model organisms that give insight into the evolutionary history of the apicomplexans. C. velia is easily cultured and has a genome project underway. However, there is an urgent need for the development of molecular toolkits and transfection systems to exploit fully the potential of these organisms. The full extent of the flagellar contribution to the apical complex, and whether the SFA-based matching mechanism is widespread among apicomplexans or an oddity of T. gondii, remain important unanswered questions. Acknowledgements Support for N.P. is provided by the University of Sydney Postdoctoral Research Fellowship scheme.
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