Current and Emerging Approaches to Studying Invasion in Apicomplexan Parasites

In this chapter, we outline the tools and techniques available to study the process of host cell invasion by apicomplexan parasites and we provide specific examples of how these methods have been used to further our understanding of apicomplexan invasive mechanisms. Throughout the chapter we focus our discussion on Toxoplasma gondii, because T. gondii is the most experimentally accessible model organism for studying apicomplexan invasion (discussed further in the section, “Toxoplasma as a Model Apicomplexan”) and more is known about invasion in T. gondii than in any other apicomplexan.

Host Cell Invasion by Apicomplexan Parasites

Host cell invasion is a complex, multi-step process that is relatively conserved among apicomplexans (see Fig. 1). T. gondii invasion begins with movement of the parasite over the surface of a host cell, driven by an actin-myosin-based motor within the parasite. This gliding motility is dependent on a solid substrate, and occurs in the absence of flagella, pseudopodia, or other traditional locomotive organelles (reviewed in ref. 2; see also Matuschewski, this volume). As the parasite glides over host cells, a trail of surface proteins and lipids is deposited in the parasite's wake.³ The conoid, a thimble-shaped cytoskeletal structure comprised of tubulin assembled into spiral ribbons,⁵ repeatedly protrudes and retracts from the apical tip of the gliding parasite (reviewed in ref. 5). Initial attachment of the parasite to the host cell is coupled to the discharge of the micronemes, apical secretory organelles that release adhesins onto the parasite surface^6,7 (see also Carruthers, this volume). A more intimate apical interaction then develops between the parasite and host cell⁸ and a second set of apical organelles, the rhoptries, discharge. Secretion from the micronemes and the rhoptries is tightly controlled, and there is some evidence that secretion from the bulb and neck regions of the rhoptries are distinct, regulated events,⁸ although the relative order of these various secretory events is not entirely clear. Very early in the process of invasion, perhaps when the micronemes and/or rhoptries are first discharged, a transient change in host cell plasma membrane conductance can be detected,⁹ suggesting a temporary, localized breach in host plasma membrane integrity. A third set of secretory organelles, the dense granules, are constitutively discharged, and are thought to function primarily in establishing and modifying the parasite's intracellular niche after invasion (reviewed in ref. 1).

Figure 1

Overview of host cell invasion by T. gondii tachyzoites. A) A tachyzoite glides over the surface of a host cell, powered by its actin-myosin-based motor. The thimble-shaped conoid at the apical tip of the parasite repeatedly protrudes and retracts. Adhesins (more...)

Active penetration of the host cell begins at the apical tip of the parasite and is thought to be dependent upon the same actin-myosin machinery that powers gliding motility, since disruption of a single myosin gene, MyoA, disrupts both processes¹⁰ and pharmacological agents that disrupt gliding motility also disrupt invasion.¹¹ Due to the duality of function of the actin-myosin motor, it is difficult to determine whether gliding motility is required for invasion; however, video-microscopic analysis of T. gondii suggests that tachyzoites may be probing, perhaps by way of conoid extension, for some specific receptor or membrane microdomain as they glide over the host cell surface before initiating invasion. Both gliding motility and invasion involve receptor-ligand interactions (see Matuschewski, this volume; Carruthers, this volume), resulting in anterior to posterior capping of surface proteins, followed by their protease-mediated shedding^7,12 (see also Dowse, this volume). Capping and shedding are thought to be responsible for the trails of surface proteins deposited at the posterior end of gliding parasites.

During penetration, a zone of tight interaction between the host and parasite plasma membranes forms at the site of parasite entry. This “moving junction” translocates from the anterior to posterior end of the parasite concurrent with penetration. Proteins secreted from the necks of the rhoptries, physically complexed with at least one microneme protein, were recently identified as components of the moving junction.^13,14 As the tachyzoite penetrates into the host cell it becomes surrounded by a parasitophorous vacuole membrane (PVM), derived from host cell plasma membrane lipids.⁹ Host plasma membrane proteins are largely excluded from the PVM as it forms, creating a compartment that does not fuse with the endolysosomal system of the host cell^15,16 (see also Sinai, this volume). The entire process of invasion takes approximately 20-25 seconds.¹⁷ Shortly after invasion, the PV relocates to a position adjacent to the host cell nucleus and becomes tightly associated with host cell mitochondria and endoplasmic reticulum.¹⁸

While the various steps in invasion, described above for T. gondii, are broadly conserved among apicomplexan parasites,^19-21 several interesting and potentially informative variations are observed in other genera. For example, the conoid appears to be absent from Plasmodium and Babesia²² (reviewed in ref. 5). Parasite reorientation, to bring the apical end in contact with the host cell, is more pronounced in other apicomplexans such as Plasmodium²³ and Cryptosporidium²¹ than it is in T. gondii. An entirely different invasive mechanism, which does not involve formation of a PVM, is employed by Plasmodium sporozoites as they migrate through host cells en route to their preferred site of replication (see Frevert, this volume). Theileria sporozoites are perhaps the most unusual of all the apicomplexans in terms of their invasive mechanisms. The invasive stages of Theileria are not motile, have no recognizable conoid or micronemes, and do not apically reorient prior to invasion. Internalization appears to be a passive process on the part of the parasite, with no role for parasite actin. However, other features of Theileria invasion are similar to those of other apicomplexans, including protease-mediated shedding of surface proteins during internalization, and formation of a PVM from the host cell plasma membrane (reviewed in ref. 24).

Assaying Host Cell Invasion

Given the importance of host cell invasion in the life cycle and pathogenesis of apicomplexan parasites, there is much interest in studying the mechanisms underlying invasion. Many different means of quantitatively assaying invasion have been developed, particularly for T. gondii, as described below.

β-Galactosidase Assay

This method measures the amount of o-nitrophenyl-β-D-galactoside (colorless) that is converted to o-nitrophenol (yellow) by parasites expressing β-galactosidase.^11,31 This assay offers the advantages of being easy, fast, and sensitive. However, in addition to the requirement to remove or kill extracellular parasites, the assay can only be used for parasites expressing β-galactosidase. Although such parasites are available for T. gondii^32,33 and Neospora,³⁴ the requirement for β-galactosidase expression may complicate further transgenic manipulation of the parasites, due to the limited number of selectable markers available (summarized in Table 1).

Table 1

Differential Fluorescent Staining

In the most commonly used assay for T. gondii invasion, parasites are labeled with different fluorochromes before and after host cell permeabilization, resulting in dual labeled extracellular parasites and singly labeled intracellular parasites (Fig. 2B, F), which are scored by fluorescence microscopy.¹¹ The use of parasites expressing YFP or GFP further simplifies the assay, by eliminating the need to permeabilize the host cells to visualize intracellular parasites.^50,75 This assay isolates invasion from other aspects of the parasite life cycle and does not require removal of extracellular parasites for accurate counting of invaded parasites. However, the assay suffers from significant field-to-field variability, which is compounded by the limited sample size that can be feasibly obtained, given the extensive hands-on time required for manual counting. An automated microscope has been used to semi-quantitatively assay invasion by differential staining in a high-throughput format.⁵⁰ In an attempt to make the differential staining assay more quantitative and reproducible, the assay was recently adapted for scoring by a laser scanning cytometer (LSC). The LSC-based assay significantly increases the number of parasites that can be counted, overcoming much of the field-to-field variability observed when the assay is scored manually. The major drawbacks to the LSC-based assay are that it requires specialized instrumentation and is not well-suited to analyzing many samples at one time.⁷⁶ However, it may be possible to adapt these assays for analysis by flow cytometry.⁷⁷

Figure 2

Small molecule inhibitors and enhancers of invasion, motility, and microneme secretion. Representative images from differential fluorescence invasion assays (B,F), trail deposition motility assays (C,G), and microneme secretion assays (D,H) of parasites (more...)

Assaying the Individual Steps of Invasion

T. gondii invasion can be readily assayed either in its entirety (using the methods described above), or as a sequence of discrete steps, each of which can be assayed independently. A recently developed protocol for synchronous invasion by T. gondii⁹⁷ further complements these assays. Parasites are allowed to settle onto host monolayers in a high potassium buffer, which inhibits parasite motility and invasion. The medium is then exchanged for low potassium medium, stimulating motility and invasion. This allows for synchronous and robust invasion over a short period of time. A temperature shift can also be used to synchronize invasion of T. gondii.⁴⁰

Assays for the individual steps of T. gondii invasion are outlined below, and similar assays for other apicomplexans, when available, are described.

Rhoptry Secretion

T. gondii tachyzoites pretreated with cytochalasinD are unable to move or invade host cells, but they retain the ability to attach to host cells and to secrete the contents of their rhoptries. The secreted rhoptry proteins can be detected as vesicular clusters (termed evacuoles) within the host cell by immunofluorescence microscopy¹²¹ (Fig. 3). The quantity, length, and protein composition of evacuoles can be assessed using different antibodies, as a means of assaying rhoptry secretion.^8,122 Evacuole-like structures are also observed within erythrocytes after Plasmodium merozoite invasion is arrested with cytochalasinB, cytochalasinD or staurosporine.^91,124,125

Figure 3

Assaying rhoptry secretion by evacuole formation. T. gondii tachyzoites treated with cytochalasinD, which inhibits actin polymerization, attach to host cells but do not invade. In this state, parasites are able to secrete some of the contents of their (more...)

Approaches to Studying Invasion

Many different experimental approaches are available for the identification and functional analysis of gene products involved in invasion. These include forward genetic screening, reverse genetic analysis, antibody inhibition, small-molecule-based approaches, alteration of the host cell, and genomic/transcriptomic/proteomic studies.

Reverse Genetics

While forward genetic screens are useful for identifying new parasite genes and proteins that function in invasion, reverse genetic approaches have been extensively used to study specific proteins postulated to function in invasion based on other criteria, such as antibody inhibition, subcellular localization, sequence homologies, or the forward genetic approaches mentioned above.

Knockout Studies

Targeted gene disruption by homologous recombination has been widely used to study gene function in T. gondii. Increasing the size of the targeting sequence seems to increase the frequency of homologous recombination: one study showed that plasmids containing 1.7, 7.6, or 15.7 kb of targeting sequence resulted in 0, 62, and 82% of the clones harboring homologously integrated plasmid DNA, respectively.¹²⁶ In T. gondii, where genomic sequence data is available, knockout vectors can be constructed using only the noncoding regions flanking the gene of interest. Utilizing coding sequence to target the knockout construct could result in expression of partial proteins, complicating interpretation of the resulting phenotype. Typically, the coding region of the targeted gene is replaced with a selectable marker (Table 1) flanked by several kb of 5' and 3' noncoding sequence. After selection, individual clones can be screened for gene disruption by Western blotting if antibodies are available.^71,137-139 Alternatively, after selection, pools of clones can be screened by PCR, utilizing one primer from within the genome and one primer from within the knockout construct to identify those parasites with the targeted disruption.⁸ In positive clones, gene disruption can be confirmed by Southern blotting, Western blotting and/or immunofluorescence microscopy.^8,71,137-139 Knockout constructs are often introduced as linearized plasmids,^138,139 to promote double crossover-allelic exchange, as opposed to single crossover events, which result in pseudodiploid formation and the possibility of expression of functional protein (Fig. 4). The transfection efficiency of circular and linear plasmids appears similar;^39,41 however, homologous recombination occurs with higher frequency when using circular plasmids, suggesting that pseudodiploid formation is the most prevalent recombination event.³⁹ Since DHFR sequence seems to promote nonhomologous integration in T. gondii (see above), inclusion of DHFR should be avoided in constructs intended for targeted knockouts.

Figure 4

Schematic illustration of recombination and integration. A) Homologous recombination at two sites (double crossover) results in allelic replacement and can be used for targeted gene disruption using circular (shown) or linear plasmids. B) Homologous recombination (more...)

Using a positive selectable marker within the targeting sequence and a negative selectable marker outside the targeting sequence selects against nonhomologous recombination and pseudodiploid formation,¹⁴¹ significantly reducing background. A similar double selection strategy is available for P. falciparum knockouts.⁶²

Attempts to generate a gene knockout could fail either because the gene is essential or because the knockout construct did not properly target the locus of interest. In order to rule out the latter, an exogenous, functional copy of the gene can be randomly integrated into the genome before attempting to knockout the endogenous locus.^142,143 Alternatively, an additional copy (mutated or functional) can be inserted by homologous recombination to create a pseudodiploid (Fig. 4B). The pseudodiploid can subsequently be resolved, under negative selection, by intrachromosomal recombination, resulting in either reconstitution of the wild type locus or formation of the mutated locus (Fig. 4C). Since vector sequence, including markers, will be removed by pseudodiploid resolution, a marker which can be used for either positive or negative selection, such as HXGPRT, is ideal for these types of experiments.¹⁴⁰ The wild type and the mutated loci should be recovered with equal frequencies; recovery of only the wild type locus suggests that the gene is essential.¹⁴³ In cases where there is a phenotype associated with a knockout, polar effects can be ruled out by complementing the knockout with an exogenous copy of the gene being studied and observing partial or complete rescue of the affected phenotype.^71,137 The choice of promoter and selectable marker should be carefully considered, since either can affect the level of expression during complementation.

Knockouts in a number of T. gondii genes have been generated, some of which result in reduced invasion. For example, MIC2 Associated Protein (M2AP) knockout parasites are significantly defective in invasion, which has been attributed to mistargeting of M2AP's partner protein, MIC2.⁷¹ Given that MIC2 itself cannot be knocked out, presumably because it is essential, this work provided valuable information on the function of both M2AP and MIC2. MIC1 knockout parasites are significantly defective in host cell invasion, but only slightly defective in mouse virulence.¹⁴⁴ In contrast, MIC3 knockout parasites are not defective in invasion and are only slightly defective in mouse virulence. Interestingly, MIC1/MIC3 double knockouts show similar levels of invasion to the MIC1 knockout but significantly reduced virulence compared to the MIC1 or MIC3 single knockouts, indicating a synergistic role for these two proteins in virulence.¹⁴⁴ A number of dense granule proteins have been knocked out,^137,139 none of which affect invasion. However, the GRA2 knockout has reduced virulence, consistent with the hypothesis that dense granule proteins are not necessary for active penetration, but are involved in PV modification and possibly immune modulation. The major glycosylphosphatidylinositol (GPI)-anchored surface proteins, SAG1 and SAG3, have both been successfully knocked out. The SAG1 knockout parasites show no defect in invasion; in fact, the knockout parasites were found to invade more quickly than wild type parasites.¹⁴⁵ In contrast, the SAG3 knockout showed reduced adhesion, invasion, and virulence, supporting the hypothesis that some members of the SAG family of proteins function as adhesins.¹³⁸

When comparing knockout parasites to wild type or complemented parasites in invasion assays, it is important to use parasites in equivalent stages of their life cycle. Parasite invasiveness diminishes progressively after release from the host cell; therefore, care must be taken to ensure that release of the two parasite populations to be compared is synchronous (Mital and Ward, unpublished). Growth assays, comparing the intracellular replication of the parasites under study, can help in the synchronization of different parasite populations.⁸

Knockouts in nonessential genes have been generated and used to study invasion in Plasmodium (e.g., refs. 82, 146-150 and reviewed in ref. 150). A useful variation on the knockoutapproach in Plasmodium has been to disrupt an essential gene in a life cycle stage that does not require the gene (e.g., the blood stages), and then analyze the invasion phenotype in the stage in which the gene is essential (e.g., mosquito salivary gland sporozoites).^78,152,153 This approach has provided a great deal of functional information about the proteins involved in sporozoite invasion (reviewed in refs. 154-157).

Conditional Knockouts

The recent development of an inducible promoter system for T. gondii¹⁰ allows the study of genes essential for invasion, and has been successfully used for four such genes (MyoA,¹⁰ TgAMA1,⁸ MIC2,¹⁵⁸ and ACP¹⁵⁹). This system is based upon an anhydrotetracycline- (Atc-) responsive transactivator protein that drives expression of genes downstream of a minimal SAG1- or SAG4-based promoter containing seven Tet operators. Expression occurs in the absence of Atc, and is repressed in the presence of Atc¹⁰ (Fig. 5). Generation of a conditional knockout is accomplished in two steps. First, parasites are transfected with the gene of interest downstream of the regulatable promoter, and clones with regulatable expression of properly localized protein are isolated. These clones are then transfected with a knockout construct to disrupt the endogenous locus of the gene of interest. The resulting parasite's sole source of the essential protein can then be turned off by addition of Atc. Conditional knockout parasites are therefore generated and isolated in the absence of Atc, and phenotypically characterized after incubation with Atc. Since phenotypic characterization is accomplished by comparing conditional knockout parasites in the presence and absence of Atc, all assays begin with the same population of parasites, alleviating the problems associated with comparing two nonequivalent populations of parasites (discussed above), provided that the knockout exhibits no growth defect.⁸

Figure 5

Generation of an AMA1 conditional knockout. A) Wild type T. gondii expressing the tetracycline-sensitive transactivator (designated “AMA1” parasites) were transfected with a plasmid containing AMA1 under control of the regulatable promoter. (more...)

The success of this approach depends critically on the expression levels of the regulatable gene relative to wild type expression; expression in the absence of Atc must be sufficiently high for parasite viability, and expression after Atc treatment must be sufficiently low to reveal a phenotype (Fig. 5A, B). The optimal expression level will vary with the particular protein being studied, and may be modulated by the number of regulatable copies integrated and the choice of promoter driving regulatable expression. Currently, two versions of the regulatable promoter are available: the SAG1-based promoter gives lower basal expression, but tighter regulation after Atc addition, compared to the SAG4-based construct (personal comm. D. Soldati). In principle, the endogenous gene could be replaced with a regulatable copy in a single step; however, the two step approach enables one to insert multiple copies of the regulatable gene, which may be necessary to achieve the required expression levels prior to disruption of the endogenous gene, and to assess whether the knockout construct can effectively target the locus of interest. Both approaches require that the endogenous gene can be effectively targeted by homologous recombination. Some residual protein may remain after Atc addition, due either to leaky expression or slow protein turnover, which should be considered when interpreting any observed phenotype, or lack thereof.

The conditional promoter system has recently been adapted for use in P. falciparum.¹⁶⁰ The development of a conditional mutagenesis system in P. berghei, based upon regulatable sitespecific recombination, presents an alternative means of constructing stage-specific knockouts; the utility of this system will increase as more stage-specific promoters are characterized.¹⁶¹

RNA-based Methods

RNA-based approaches to diminishing, rather than abolishing, expression of a protein are useful for genes that cannot be studied by conditional knockout, either because the required expression levels do not fall within the range of the available conditional promoters, or because the gene cannot be effectively targeted for disruption. In T. gondii, RNA-based techniques such as antisense, double stranded RNA interference (RNAi), and delta ribozymes have been successfully used in a limited number of laboratories to modulate gene expression.^162-166 All of these techniques rely on short sequence recognition. The sequence must therefore be carefully chosen to assure knockdown of a specific gene, especially if that gene is part of a gene family or shares significant sequence with other proteins (although this can be an advantage if knockdown of a multigene family is desired.) Optimization of sequence and RNA expression level may be required to achieve a threshold of knockdown that maintains viability of the parasite but results in an invasion phenotype.

As yet, only the antisense RNA technique has been used to study invasion-related genes. Antisense RNA was used to decrease expression of the rhoptry protein ROP2, which was thought to be essential for invasion since previous knockout attempts were unsuccessful.¹⁶⁴ A significant decrease in invasion, among other defects, was seen in ROP2-deficient parasites. Antisense RNAs have also been used to study the T. gondii dense granule protein NTPase, which is expressed as two isoforms from different loci¹⁶⁷ and is thought to be essential.¹⁶⁵ Despite these apparent successes, antisense methods have not yet gained widespread use in T. gondii due to difficulties with reproducibility. Antisense methods have also been used in Plasmodium.^168,169

Homologues of many of the genes involved in RNAi have been found in T. gondii,¹⁷⁰ but not Plasmodium.¹⁷¹ However, RNAi has been reported to work in both Plasmodium^172-174 and T. gondii.¹⁶² RNAi has been used to show that decreased expression of two independent lactate dehydrogenase genes in T. gondii results in reduced differentiation and virulence.¹⁷⁵ Double-stranded RNA has also been used to knockdown a T. gondii immunophilin.¹⁷⁶ Since extensive controls are needed to unambiguously attribute downregulation to the RNAi pathway, there remains some uncertainty about whether the effects observed in these studies result from true RNAi.¹⁷⁰

A recent report on the use of catalytically active delta ribozymes to bind and cleave specific targets¹⁷⁷ indicates that this may also be a useful technique for studying essential genes.

Transgene Expression

Intra- and inter-species expression of wild type or mutated transgenes has been useful for studying apicomplexan invasion-related proteins. Care must be taken when analyzing data from exogenous expression to ensure that expression levels, post-translational modifications, and trafficking are comparable in the exogenous and native proteins. Additionally, codon bias may need to be considered; whereas T. gondii genes have been expressed in a variety of systems (e.g., refs. 34, 178, 179), codon bias in the A/T rich genome of P. falciparum¹⁸⁰ can cause difficult, though not insurmountable problems for heterologous expression.^181-184

Expression of various mutated and truncated T. gondii microneme proteins in wild type T. gondii has proven to be a useful approach for elucidating the role of these proteins in invasion and for defining their functional domains. For example, exogenous expression of mutated forms of MIC2 in T. gondii helped to elucidate MIC2's role as an adhesin and to define the mechanisms governing microneme protein cleavage (reviewed in ref. 185). Flow cytometry can be used to identify mutations that cannot be tolerated by the parasite.¹⁸⁶

Expression of T. gondii proteins in other apicomplexans, or vice-versa, has provided insights into invasion by both the donor and recipient species. For example, functional homology between the cytoplasmic tails of Toxoplasma MIC2 and Plasmodium TRAP, microneme proteins thought to function in motility and invasion, was demonstrated by the ability of the cytoplasmic tail of Toxoplasma MIC2 to rescue the motility and invasion defects caused by deletion of the TRAP cytoplasmic tail in Plasmodium sporozoites.¹⁸⁷ Proper expression and localization of a number of Toxoplasma proteins in the related apicomplexan Neospora^34,65,178 indicate that this may also be a useful system to study functional homology and elucidate protein function. Inter-species expression of proteins will be particularly useful for studying protein function and invasion in organisms that are difficult to culture and/or for which molecular genetic tools are not yet available, such as Cryptosporidium.¹⁸⁸ Expression of Plasmodium proteins in other species of Plasmodium has proven to be a useful way to study the function of secreted and cell surface proteins and to identify the parasite proteins responsible for host range specificity (Fig. 6A, C).^189,190 Finally, expression of apicomplexan genes in both yeast¹⁹⁵ and mammalian cells have been useful for determining the role of purported adhesins in host cell attachment (Fig. 6B).^{118,191-193,196}

Figure 6

Inter-species approaches to studying invasion. A) Treatment of P. falciparum merozoites with an antibody against P. falciparum AMA1 inhibits invasion of human erythrocytes (D10: white bar = untreated; grey bar = antibody treated). The expression of P. (more...)

Small-Molecule-based Approaches

The most common way to use small molecules to study a process such as invasion is to determine whether specific pharmacological agents with known targets in other systems affect the process of interest, thereby implicating the known target in the process. For example, the first evidence implicating actin in the process of invasion, both in T. gondii^115,209,210 and Plasmodium¹²⁵ was the inhibition of invasion by cytochalasinB or D. Similarly, inhibition of microneme secretion by the intracellular calcium chelator BAPTA-AM, together with stimulation of microneme secretion by the calcium ionophore A23187, strongly suggested a role for intracellular calcium in microneme secretion.^31,211

A more general way to use small molecules, which is not limited to compounds with predefined targets, is “phenotype-based” small molecule screening.²¹² In such an approach, libraries of structurally diverse small molecules are screened for those that generate a particular phenotype; these compounds are then used to directly identify the target(s) responsible for generating the phenotype (Fig. 7). The approach is essentially the pharmacological analog of classical forward genetics (compounds that disrupt protein function being analogous to mutations); however, in the small molecule approach, perturbation of protein function is under the investigator's control rather than being permanently encoded in the mutant's genome. When applied to invasion, this circumvents the problem of invasion mutants being nonviable. In a recent screen of 12160 small molecules, 24 novel inhibitors and 6 enhancers of T. gondii invasion were identified,⁵⁰ most of which were fully reversible. Different compounds had distinctly different effects on microneme secretion, motility, and/or conoid extension.

Figure 7

Overview of phenotype- vs. target-based small molecule approaches to studying biological processes. See text for details. Reproduced from: Ward GE et al. Cell Microbiol 4(8):471-482; ©2002, with permission from Blackwell Publishing.

The most difficult part of any small molecule-based screening project is target identification. A number of biochemical and genetic approaches are available for target identification and have been recently reviewed.^212-215 “Activity-based probes,” which can be designed to covalently modify specific subsets of enzymatic targets, can facilitate target identification (reviewed in ref. 113). Although such probes sample only a subset of the proteome, if this subset is of interest (e.g., a particular family of proteases), the selectivity is an advantage, and the ability of the probes to covalently modify their targets can greatly facilitate target identification. In all cases, the identified target must be independently validated as the relevant target in vivo.^212-215

An alternative way to use small molecules is to screen large collections of structurally diverse small molecules for compounds that affect the activity of an individual recombinant or purified protein (“target-based” screening; Fig. 7), rather than causing a complex, cell-based phenotype. Once a small molecule inhibitor of that protein has been identified, it can be used on whole cells to determine the function of the protein within the context of the cell (the pharmacological analog of reverse genetics). Target-based screening has been used to identify novel inhibitors of T. gondii dense granule NTPases²¹⁶ and P. falciparum dihydroorotate dehydrogenase.²¹⁷ One clear benefit of taking a small molecule approach is that it may generate not only new tools for studying invasion, but also new potential lead compounds for drug development.

Toxoplasma as a Model Apicomplexan

The overall process of invasion appears to be relatively well conserved among apicomplexan parasites, even if the particular molecules mediating the process vary. For example, the individual microneme proteins of Toxoplasma and Plasmodium are different, but many are organized along a similar theme, i.e., modules of adhesive motifs.²⁵⁴ In fact, portions of some microneme proteins are functionally interchangeable between Toxoplasma and Plasmodium, despite limited sequence identity.¹⁸⁷ These and other examples^8,50,157,205 clearly demonstrate that findings from one species of apicomplexan parasite can often be extended to others.

One of the main reasons why T. gondii has become a model for studies of apicomplexan invasion (reviewed in ref. 255) is its amenability to molecular genetic manipulation (see previous section on “Approaches to Studying Invasion”). Several selectable markers are available, exogenous DNA integrates with high frequency and is readily expressed, and a variety of approaches can be used for gene knockouts, conditional gene expression, and chemical or insertional mutagenesis (Table 1). The genome is completely sequenced and readily accessible (ToxoDB.org). In addition, the T. gondii tachyzoite (2 × 7 μm) is considerably bigger than, e.g., the Plasmodium merozoite (1 × 1 μm), facilitating subcellular localization of proteins and a variety of other cell biological techniques. T. gondii is easily cultured, and the ability of T. gondii tachyzoites to invade virtually any nucleated mammalian cell creates powerful opportunities for studying the host cell requirements for invasion.^218,219 As described above, assays have been developed that allow each step of T. gondii invasion to be examined in isolation, and a well-developed mouse model is available for in vivo studies. The ability to express non-T. gondii genes in T. gondii^188,256,257 (see above) also makes it an excellent model for studying genes from less experimentally tractable parasites.

Outstanding Questions

While a great deal of progress has been made in recent years in our understanding of the invasive mechanisms of apicomplexan parasites, many important questions remain. These include:

Conclusion

Given the powerful combination of experimental tools currently available, it is our hope that we will soon more fully understand the molecular mechanisms of host cell invasion by apicomplexan parasites. Our efforts can then be directed towards developing effective ways of blocking these processes, as a means of preventing and treating the devastating diseases caused by these important human pathogens.

Acknowledgements

We thank Vern Carruthers, Chetan Chitnis, Stacey Gilk, and members of our laboratory for helpful comments on the manuscript. We also gratefully acknowledge the support of the Burroughs-Wellcome Fund and Public Health Service grants AI054961 and AI063276.

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