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Keywords:

  • actin polymerization;
  • actomyosin motor;
  • Apicomplexa;
  • gliding motility;
  • host cell invasion;
  • malaria;
  • microfilaments;
  • Plasmodium

Abstract

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References

Efficient and rapid host cell invasion is a prerequisite for an intracellular parasitic life style. Pathogens typically induce receptor-mediated endocytosis and hijack the force-transducing system of a host cell to gain access to a replication-competent niche. In striking contrast, apicomplexan parasites such as Plasmodium, the causative agent of malaria, and the human and animal pathogens Toxoplasma and Cryptosporidium employ their own actomyosin motor machinery to propel themselves into prospective host cells. Understanding the regulation and dynamics of actin-based motility of these parasites is therefore central to understanding their pathogenesis. The parasite genomes harbour surprisingly few potential actin-regulatory proteins indicating that a basic repertoire meets the requirements to regulate actin dynamics. In this article, we summarize our current knowledge of Plasmodium microfilament dynamics and describe its potential players.


The central role of microfilaments in the motility of apicomplexan parasites

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References

Malaria is an infectious disease caused by facultative intracellular parasites of the genus Plasmodium. These parasites belong to the phylum Apicomplexa that also includes ubiquitous human and animal parasites, such as Toxoplasma and Cryptosporidium. All apicomplexan parasites form specialized invasive stages that cover a range of different host cell types but share a unique mechanism of motility and host cell entry (1). Notably, parasite locomotion (i) does not involve motor organelles, (ii) is actin dependent, and (iii) occurs at a speed comparable to that of in vitro-reconstituted actomyosin interaction (several μm/second). As we understand it to date, invasins of the thrombospondin-related anonymous protein (TRAP) family recognize host cell surface components and transmit this extracellular recognition event to the parasite cytoplasm by binding to actin filaments through tetramers of aldolase (2,3). Unconventional class XIV myosins that are immobilized to the inner membrane complex, an additional microtubule-supported membrane layer beneath the parasite plasma membrane, move the actin–aldolase–TRAP complexes toward the posterior end of the parasite, thereby driving it forward [Figure 1 (4)]. Thus, rather than inducing uptake by their host cell, the invasive parasite stages, powered by their own actomyosin motor machinery, actively invade the cells.

image

Figure 1. Model of the regulation of microfilament dynamics in Apicomplexa. Actomyosin-based gliding motility and host cell invasion of the parasites is dependent on the existence of stable actin polymers of defined length and regulated turnover thereof. This process requires three principal regulatory steps: (i) actin polymerization at the apical end, (ii) rearward movement of filamentous actin-receptor units, and (iii) depolymerization of actin filaments. A) Assembly of actin polymers. Near the apical pole of the parasite, actin forms polymers with the help of polymerization machineries, such as formin or functional equivalents of Arp2/3, and are stabilized by factors such as capping protein. The interactions with actin-binding proteins may be dependent on the phosporylation state of the actin-bound nucleotide. This intrinsic actin ATPase activity may also account for differences in the biochemistry of actins from various parasites. B) Engagement of stabilized actin polymers in motility (grey arrow). Short actin polymers bind transmembrane receptors through aldolase tetramers or additional linkers and myosin heads fixed to the inner membrane complex. Actin scaffolds may thus integrate the forces needed to drive the parasite forward and pass them on to the surface receptors. C) Disassembly of the motor machinery and F-actin depolymerization. Surface receptors are released from host cell components by the action of rhomboid proteases (55). Actin scaffolds need to be regenerated which likely engages the activities of actin monomer sequestering proteins and nucleotide exchange factors like ADF, CAPlike and profilin. In analogy to other systems, monomers are shuttled to profilin to be kept in a polymerizable form. However, in contrast to other systems, the intrinsic instability of parasite F-actin may not require the action of F-actin depolymerizing proteins.

Despite the central importance of parasite actin filaments to the motor machinery (5,6), parasite microfilaments can be visualized neither by electron microscopy nor using fluorescent derivatives of the filamentous actin (F-actin)-binding toxin, phalloidin (7,8). Abnormal actin polymerization can be induced at the apical end of motile parasites with jasplakinolide (9,10). Such aberrant microfilaments impair host cell invasion (10) and change both velocity and orientation of gliding T. gondii tachyzoites (11), the pathogenic stages that cause toxoplasmosis in immunocompromized individuals. This finding suggests that actin polymerization is the rate-limiting factor for gliding motility and host cell entry.

Apparently, the dynamics of the apicomplexan microfilament system differ fundamentally from the well-understood regulated turnover of actin in mammalian cells. The properties of apicomplexan actin-binding proteins characterized until now cannot explain these unusual actin dynamics, and it remains unknown how microfilaments are regulated and how actomyosin force generation works in these parasites. However, with several complete parasite genome sequences at hand and several more being close to finished, our understanding of actomyosin motility and microfilament regulation in Apicomplexa can gain by an inventory of the genes that are likely to be involved.

Apicomplexan parasites: same but different

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References

Protozoans of the phylum Apicomplexa are divergent organisms with a common mode of motility and similar general life cycles. It is therefore tempting to apply insights from one parasite species to the rest of the group. However, important differences exist among members of the phylum. For example, Plasmodium species express two conventional actin isoforms (12,13), while the other taxa within this phylum have only one (Table 1). Similarly, both parasites possess individual sets of actin-related proteins (Arp) (14) and actin-binding proteins (15).

Table 1.  Actins and potential actin-binding proteins in Plasmodium, Toxoplasma and Cryptosporidiuma
  • a

    Note that the table contains predicted proteins with homology to actin and actin-binding proteins. Predicted molecular weights of P. falciparum proteins are shown in brackets and may vary slightly between different genera and species. Accession numbers are according to www.plasmodb.org, www.toxodb.org and www.crytodb.org. Indicated in brackets after each accession number is the extent of sequence identity compared with the respective P. falciparum ortholog. Abbreviations: 14-3-3, 14-3-3 phosphoprotein binding family; ACT, actin; ADF, actin depolymerizing factor; ALP1, actin-like protein-1; CAPlike, adenylate cyclase associated protein like; CAP, C-terminal extended -sheet domain of cyclase associated proteins; CP, capping protein; FH2, formin homology-2 domain; FLP, formin-like protein; PROF, profilin; WD40, WD40 repeats.

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Apicomplexan Microfilaments: Get Shorty

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References

Remarkably, the main Plasmodium actin isoform (PfACT1; PFL2215w) polymerizes inefficiently alone (16). This limited intrinsic polymerizability could only be overcome by combined addition of two non-physiological components, gelsolin and phalloidin. The PfACT1 polymers stabilized in that manner appeared fragmented in the fluorescence microscope. Importantly, these actin polymers were capable of binding fluorescent phalloidin derivatives suggesting that the failure to visualize parasite microfilaments is due to low steady-state levels of unusually short actin polymers. This conjecture is corroborated by recent findings showing that harvested functional actin polymers were of extremely short length [around 100 nm, corresponding to an average of 50 protomers only (17)]. Similarly, T. gondii actin forms filaments with a short persistence length, albeit with a low critical polymerization concentration (18). Thus, apicomplexan actin polymers have gone undetected for so long probably simply because they are very short. As has recently been pointed out, these unexpected findings imply a unique mode of microfilament function in these parasites: Formation of short actin polymers may be transiently induced by regulatory proteins, and the F-actin stubs thus formed might act as scaffolding units, bringing together surface receptors and motor molecules [Figure 1 (19)].

Parasite actin-binding proteins: a minimal microfilament regulatory system

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References

The actin stubs model for parasite actomyosin motility shown in Figure 1 can explain the observations that were puzzling in the light of a conventional microfilament system. But what are the components needed to make motility based on F-actin stubs work? Homology searches of apicomplexan genomes for microfilament proteins return a strikingly low number of orthologs of proteins that one might expect in highly motile cells expressing one or two different actin isoforms (Table 1). Many protein families that are considered key actin regulators in yeast and higher eukaryotes are absent in Apicomplexa, and where orthologs do exist they often display prominent heterologies. For example, in the expanding leading edge of motile mammalian cells, the creation of transient-free barbed ends that support actin polymerization is associated with the severing activity of gelsolin, while filament nucleation and branching is mediated by the Arp2/3 complex (20). Neither gelsolins nor components of the Arp2/3 complex are encoded in apicomplexan genomes (12,14). Other examples of actin-modulating proteins apparently missing in Apicomplexa include most families of filament cappers, filament cross-linking proteins like α-actinin or fimbrin, actin depolymerizing factor (ADF)-interacting and F-actin depolymerizing capping protein (CP), actin interacting protein-1 (Aip1) and regulatory classes like Mena/Ena/VASP proteins, WASP homology (WH)-1-domain containing proteins (verprolin) and WH2-domain containing proteins [Wiskott-Aldrich syndrome protein (WASP)/WASP family verprolin homology protein (WAVE)/suppressor of cAMP receptor (Scar)].

Monomer-binding proteins

Actin depolymerizing factor/cofilins are among the most important proteins catalyzing microfilament turnover. They serve as actin polymers and enhance the rate of dissociation at the pointed ends (21). In contrast to Toxoplasma and Cryptosporidium, Plasmodium species express two ADFs, one of which has recently been characterized. The Plasmodium ortholog (PfADF1; PFE0165w) is abundantly expressed in all motile stages and binds monomeric actin with a preference for ADP-bound over ATP-bound monomers, but does not interact with actin polymers (22). Moreover, while other ADFs inhibit nucleotide exchange on monomeric actin, this essential Plasmodium ortholog stimulates nucleotide exchange. Thus, the biochemical properties of recombinant PfADF1 suggest that the protein may spur F-actin formation by interacting with ADP-actin monomers in order to generate polymerizable ATP-actin monomers.

The PfADF1 as well as the ADF sequences of Cryptosporidium and Toxoplasma all lack the established actin-polymer-binding motifs (22,23). In the light of these new findings, the reported F-actin-binding activity of the recombinant Toxoplasma protein (24) may need to be studied in greater detail. Based on structural models, it has been suggested that non-homologous replacements in the apicomplexan actin sequences may explain the intrinsic instability of parasite microfilaments (16,18). In such a scenario, the filament depolymerizing activity of ADF/cofilins may not be required. Notably, the second Plasmodium ADF isoform, PfADF2 (PF13_0326), resembles conventional ADFs and apparently contains the F-actin-binding sites that are lacking in PfADF1. However, this protein appears to serve a specialized function that may be restricted to life cycle stages that include neither the pathogenic merozoites nor the transmissible sporozoites (22).

The regulation of Plasmodium ADF activity remains to be characterized. Typically, ADF activity is under tight regulation by a phosphoregulatory switch mechanism: Phosphorylation/dephosphorylation at an amino-terminal serine results in deactivation/activation of ADF and, as a consequence, in F-actin stability or F-actin disassembly and severing, respectively (25). Apicomplexan ADFs all retain a serine in position 3, but the parasite genomes encode none of the two ADF-selective kinase families that phosphorylate and inactivate ADF, LIM kinases (LIMK) (26) and TES kinases (TESK) (27). However, with 99 putative protein kinases in the parasite genome (28), phosphorylation may well be exerted by functional equivalents of LIMK and TESK, which remain to be identified. In support of the existence of an ADF kinase, a paralog of 14-3-3ξ, a class of proteins that stabilizes phosphorylated, inactive ADF (29) is present. Of the corresponding ADF-specific phosphatases, Slingshot phosphatase (30) is missing while a potential paralog of chronophin (31), a member of the haloacid dehalogenase family of phosphatases, is present, at least in the genomes of Plasmodium and Toxoplasma species.

The major actin-sequestering protein in non-muscle cells is profilin, which keeps actin monomers in an unpolymerized yet polymerization-competent state (32). Polymerization from profilin–actin complexes is mediated by WASP homology domains, apparently lacking in the phylum Apicomplexa, and by formin proteins (discussed below). Apicomplexan profilins are unusually long, containing at least an extra stretch of >25 amino acids inserted into the consensus at a site that, by structure-based sequence comparison, appears to be outside the actin- and polyproline-binding sites. The recombinant Plasmodium protein binds both monomeric actin and polyproline (H.S., unpublished data) showing that this Apicomplexa-specific sequence does not disturb interaction with these two ligands. Therefore, as profilin is also implicated in other functions, this sequence may relate to aspects other than actin regulation. Apicomplexan parasites probably express a single profilin isoform, which may be expected to exert vital functions, possibly also during parasite locomotion and host cell entry. We expect that in the absence of readily polymerizable actin, the role of profilin in formin-mediated polymerization will be crucial for the parasites, while the monomer sequestering function will be less significant. Likewise, the role of profilin as an actin nucleotide exchange factor will be less important in the presence of an ADF isoform with similar properties.

Adenylate cyclase-associated protein [CAP (33)] is a ubiquitous eukaryotic protein that binds monomeric ADP-actin. Interestingly, apicomplexan CAP-like orthologs exist that correspond only to the C-terminal portion of conventional CAPs, thus comprising the actin-binding domain, while lacking the regulatory adenylate cyclase-interacting domain. The latter domain has been shown to also mediate a ternary interaction with F-actin and ADF (34), a function that may be redundant in the absence of an F-actin-binding ADF in motile Plasmodium stages (22). The CAP-like ortholog from Cryptosporidium parvum displays the same twin-lobed extended β-sheet structure as the actin-binding domain of the yeast CAP ortholog, Srv2 (35,36).

A striking example for divergence of protein function within this phylum is the actin modulator toxofilin (15,37) from Toxoplasma, which lacks altogether in Plasmodium, Cryptosporidium and Theileria. Toxofilin sequesters actin monomers and caps filament ends, and green fluorescent protein-toxofilin impedes actin turnover in mammalian non-muscle cells. Toxofilin contains a cleavable signal peptide and was recently found in secretory organelles using a proteomics approach (38). Thus, toxofilin is likely secreted by the parasite and may perform regulatory functions in the host cell and possibly also in the parasite.

Actin polymerization machineries

Formin/diaphanous family proteins trigger monomer addition from profilin–actin complexes at the barbed end while capping that end (39). The barbed-end capping activity is mediated by the ring-like structures of the formin homology-2 (FH2) domains, while the profilin-binding activity resides in the adjacent FH1 domain. Notably, the apicomplexan genomes encode two to three FH2-domain containing proteins indicating that this mode of regulation of profilin-mediated actin polymerization may be conserved. Apart from the FH2 domain, Plasmodium formin-like protein-1 (FLP1; PFL0925w) also contains a rudimentary FH1 domain, whereas FLP2 (PFE1545c) contains two oligo-proline stretches upstream of the FH2 domain. These sequences may well be sufficient for recruiting profilin–actin complexes (40). Other domains typically found in formin/diaphanous proteins, such as the Rho GTPase-binding domain, the self-regulatory Diaphanous autoinhibitory domain (DAD) and the dimerization motif (39) cannot be found in the parasite orthologs, posing the questions of whether and how they dimerize and how they are regulated. Although apicomplexan FH2-containing proteins remain uncharacterized so far, expression profiling revealed a characteristic upregulation of PfFLP2 in invasive stages (41) supporting a role in transient rapid formation of microfilaments during parasite locomotion.

The Arp2/3 complex is an important filament nucleator in the leading lamella of motile cells. Its components Arp2 and Arp3 form a polymer end template on which actin monomers can add to build a filament (42). None of the principal Arp2/3 complex components is encoded in the genomes of Apicomplexa; however, there are a number of actin-related sequences in apicomplexan genomes the evolutionary relationship of which has been analyzed recently (14). These authors suggest that actin-like protein-1 (ALP1; Pf11_0114), which is most similar in sequence to Arp2 and Arp3, may perform Arp2/3-like functions. This is an interesting hypothesis especially in the light of poorly polymerizing Plasmodium actin. It evokes the questions of whether ALP1 would need to form a homo- or a hetero-dimer to nucleate actin polymers, and whether a putative ALP1 nucleator complex would need additional regulatory components. Also other Arps or ALPs might be able to interact with only one end of an actin polymer to nucleate or restrict polymer growth. At least several of the nine actin-related proteins are likely involved in the regulation of actin turnover. Of these, Arp1 (PFA0190c) may be part of the dynactin complex as in other organisms (14).

Actin filament-binding proteins

Filamentous actin capping is a means of preventing uncontrolled filament elongation, while simultaneously stabilizing filament nuclei for elongation at the free end. Given the low intrinsic stability of parasite F-actin (16–18), this nuclei stabilizing function is likely of particular importance for Apicomplexa.

In the absence of gelsolin genes, the putative barbed-end capping protein up-regulated in infective sporozoites-17 (UIS17) PfCPβ (PFE0880c), with homology to the β-subunit of mammalian CP, and its corresponding partner PfCPα (PFE1420w) are candidates for microfilament nucleating and stabilizing proteins in Plasmodium. The UIS17/PfCPβ was first identified in a screen for genes that are specifically upregulated in motile infectious sporozoites (43). It is still unclear whether these CPs are identical with the members of a previously identified heterotrimeric protein complex with F-actin-binding and capping activity, which also contained hsp 70 (44). Notably, CP is not necessarily implicated in actin regulation, as it is also a candidate component of the dynactin complex, the additional components of which are well conserved in apicomplexan genomes (14).

Coronins are F-actin-binding and bundling proteins implemented in microfilament regulation, actin–tubulin cross-regulation and microfilament-linked membrane trafficking (45). A coronin gene (PFL2460w) has been identified in Plasmodium and the protein shown to be associated with filamentous actin (46). This coronin does not retain the extremely basic N-terminal signature sequence, but it contains three WD40 repeats and a C-terminal coiled-coil region, thereby resembling the short orthologs that have been linked to actin regulation. The Plasmodium ortholog shares sequence similarity with the C-terminal part of yeast coronin, but lacks the MAP1B-homology region implicated in microtubule binding. It also lacks PST-rich sequences found, for example, in human coronin-7, a protein that does not interact with actin and is linked to Golgi trafficking.

Apicomplexa possess unconventional myosins with considerable divergence from the myosins of other species (47). The Plasmodium genome encodes probably six different myosins (48) which all belong to the phylum-specific class XIV, with single heads and relatively short tails. The Plasmodium myosins remain largely uncharacterized except for Myosin A (MyoA; Pf13_0233), which, like its paralog TgMyoA in Toxoplasma (49,50), is likely the motor that drives parasites forward and powers invasion of host cells. The PfMyoA localizes to the periphery of invasive stages (51,52) and is fixed to the inner membrane complex through its binding partner, Myosin A tail domain interacting protein (MTIP) [PFL2225w (53)], an interaction that has been reconstituted in vitro to produce actomyosin motility (54). Apart from their role in force generation, myosins may induce actin filament formation and participate in F-actin turnover regulation. Therefore, in the presence of only a few classes of actin regulators, it will be interesting to test if the apicomplexan myosins also classify as regulators of actin turnover. An attractive hypothesis is that the otherwise unstable Plasmodium actin polymer stubs assemble at the tip of myosin heads (Figure 1), a stabilizing interaction that would bring them into their correct cellular context.

Outlook

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References

The molecular differences between host and parasite actins and actin-modulating proteins are of great medical interest because they may suggest regulatory mechanisms for specific activation of the parasite microfilament system. Such mechanisms could reveal potential targets for drug therapy of malaria, one of the three major global infectious diseases identified in the world health organization priority program.

Infectious bacteria like Shigella and Listeria and viruses like Vaccinia hijack the actin cytoskeleton of their host cells for their own translocation. In the past decade, the study of these microbial pathogens led to the identification of several new classes of proteins, which were subsequently realized to be central to the regulation of cytoskeletal dynamics in all cell types. In analogy, the peculiar gliding motility of protozoan parasites with their minimalistic set of actin regulatory proteins may contain answers to fundamental questions.

References

  1. Top of page
  2. Abstract
  3. The central role of microfilaments in the motility of apicomplexan parasites
  4. Apicomplexan parasites: same but different
  5. Apicomplexan Microfilaments: Get Shorty
  6. Parasite actin-binding proteins: a minimal microfilament regulatory system
  7. Outlook
  8. Acknowledgments
  9. References