Molecular Characterization of a New Babesia bovis Thrombospondin-Related Anonymous Protein (BbTRAP2)

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Abstract

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

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Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa

Kelly Brayton, Audrey Lau, David R. Herndon … (2007)

Introduction Babesiosis is a tick-borne, hemoprotozoan disease enzootic in ruminants in most sub-temperate and tropical areas of the world (reviewed in [1]). It is recognized as an emerging zoonotic disease of humans, particularly in immunocompromised individuals [2], and is of historical significance as the first protozoan agent recognized to be arthropod transmitted [3]. With no widely available vaccine and a nearly global distribution, babesiosis is one of the most important arthropod-transmitted diseases of cattle, with over half of the world's cattle population at risk [4]. Live attenuated vaccines are used for the control of babesiosis in many parts of the world, but rely on region-specific attenuated strains for which vaccine breakthrough is not uncommon (reviewed in [5]). Due to the blood-based production of these attenuated vaccines and the possibility of reversion to virulence with tick passage, they are not licensed in the US. The consequences of a disease outbreak in a naïve cattle population with no available vaccine would be catastrophic. Babesia, the causative agent of babesiosis, is in the order Piroplasmida within the phylum Apicomplexa [6]. Similar to other members of this phylum, such as the phylogenetically closely positioned Theileria and its distant cousin, Plasmodium, Babesia undergoes a complex life cycle that involves both vector and mammalian hosts. In contrast to Plasmodium, for which Anopheles mosquitoes vector transmission, Theileria and Babesia are transmitted via tick vectors. For all three hemoprotozoans, sporozoites are injected into the blood stream of the mammalian host and it is at this stage where the life cycle of Babesia differs from that of Theileria and Plasmodium. For Theileria, infection leads first to lymphocytic stages followed after schizogony by intraerythrocytic development [7]. In plasmodial infection, the sporozoite first infects hepatocytes in which the stage infecting the erythrocytes is produced. In contrast, babesial infection with sporozoites leads directly to infection of erythrocytes. Once inside an erythrocyte, both Theileria and Babesia are found in the cytoplasm while Plasmodium resides in a parasitophorous vacuole. In spite of the differences in the mammalian cell types that the parasites invade, the hallmarks of a B. bovis–induced clinical syndrome in cattle, including severe anemia, capillary sequestration of infected erythrocytes, abortion, and a neurologic syndrome, are remarkably similar to human malaria caused by Plasmodium falciparum [8,9]. Whether the mechanisms leading to these clinical features are unique or are shared between these two related hemoprotozoans is unknown. Complete apicomplexan genome sequences for T. parva, T. annulata, and P. falciparum have been reported [7,10,11]. Comparisons of these genomes revealed that only approximately 30%–38% of the predicted proteins could be assigned a function, suggesting that the majority of the proteins for these organisms are novel [10,11]. Data from the genome sequences demonstrate many differences between Plasmodium and Theileria, such as the number of rRNA units and their developmental regulation, the lack of key enzymes in certain metabolic pathways, lengths of intergenic regions, gene density, and intron distribution. The genome sequence of the virulent, tick-transmissible Texas T2Bo isolate of B. bovis, reported here, will allow for an even more comprehensive, genome-wide comparison of this triad of important vector-borne apicomplexan hemoprotozoa, and can be used to identify genes that play common and species-specific roles in apicomplexan biology. Furthermore, insight from such comparisons may improve our ability to design potential prophylactic and therapeutic drug targets. Results/Discussion Genome Structure and Sequence Assembly of whole genome shotgun sequence data of the Texas T2Bo isolate of B. bovis indicates that the parasite contains four chromosomes, confirming previous results from pulse field gel electrophoresis [12,13]. Chromosome 1, the smallest of the four chromosomes, contains a large physical gap flanked by two large contigs (821,816 bp and 285,379 bp in length). The gap is estimated to be 150 kbp by pulse field gel electrophoresis (unpublished data) and contains five contigs that vary in size from 12 kbp to 28 kbp, with the order of the contigs in the gap unknown. Chromosomes 2 and 3 were fully sequenced and are 1,729,419 and 2,593,321 bp in length, respectively. Chromosome 4 contains an assembly gap that has not been unambiguously resolved; a 1,149 bp contig separates two contigs of 827,912 bp and 1,794,700 bp. Thus, the nuclear genome of B. bovis consists of four chromosomes of 2.62, 2.59, 1.73, and ∼1.25 Mbp in length. At 8.2 Mbp in size, the genome of B. bovis is similar in size to that of T. parva (8.3 Mbp) [10] and T. annulata (8.35 Mbp) [7], the smallest apicomplexan genomes sequenced to date (Table 1). Table 1 Genome Characteristics of B. bovis, T. parva, and P. falciparum Each B. bovis chromosome contains an A+T-rich region ∼3 kbp in length presumed to be the centromere (Figure 1) based on features similar to those described for the putative centromere on P. falciparum chromosome 3 [14]. Three of the chromosomes are acrocentric, while chromosome 4 is submetacentric. The organization of telomeres and sub-telomeric regions resembles that seen in Theileria [7,10], as protein coding genes are found within 2–3 kbp of the end of CCCTA3–4 telomeric repeat sequences. The B. bovis genome contains three rRNA operons, two on chromosome 3 and one on chromosome 4, and 44 tRNA genes distributed across all four chromosomes. A total of 3,671 nuclear protein coding genes are predicted in the B. bovis assembled sequence data. In addition to the nuclear genome, the parasite contains two A+T-rich extra-chromosomal genomes: a circular 33 kbp apicoplast genome and a linear ∼6 kbp mitochondrial genome (Table 1), described below. Figure 1 Representation of B. bovis Centromeres, ves1, and smorf Genes Each chromosome is represented by a black line, with the chromosome number shown on the left. Centromeres are depicted as black dots. ves1 loci are depicted as colored boxes, and the number of ves1 and smorf genes in each locus is shown above or below the colored boxes, respectively. Red and blue boxes indicate the presence of at least one ves1α/ves1β or ves1α/ves1α pair, respectively, within the cluster. Metabolic Potential and Membrane Transporters A series of in silico metabolic pathways for B. bovis were reconstructed from 248 proteins assigned an EC number, including glycolysis, the tricarboxylic acid cycle and oxidative phosphorylation, de novo pyrimidine biosynthesis, glycerolipid and glycerophospholipid metabolism, the pentose phosphate pathway, and nucleotide interconversion (Figure 2). Notably, a number of major pathways appear to be lacking in the parasite, including gluconeogenesis, shikimic acid synthesis, fatty acid oxidation, the urea cycle, purine base salvage and folate, polyamine, type II fatty acid, and de novo purine, heme, and amino acid biosyntheses. Although heme biosynthesis activity present in P. falciparum is predicted to be absent in B. bovis, it does encode delta-aminolevulinic acid dehydratase (BBOV_II001120), which catalyzes the second step in heme biosynthesis. Figure 2 Comparison of Major Metabolic Pathways in B. bovis, T. parva, and P. falciparum Solid arrows indicate single step enzymatic reactions, dashed lines indicate multi-step reactions, and dotted lines indicate incomplete or unknown pathways. Inhibitory drugs are indicated with red arrows. Glucose is assumed to be the major carbon and energy source. Non-functional pathways in P. falciparum, T. parva, and B. bovis are shown in boxes with a red X. Pathways denoted with blue text are present in P. falciparum, T. parva, and B. bovis. Pathways denoted with green text are present in P. falciparum and T. parva but not in B. bovis, and pathways denoted with red text are exclusively found in P. falciparum. The predicted metabolic profile of B. bovis is more similar to that of Theileria [7,10] than to that of P. falciparum [11]. Like Theileria, B. bovis does not appear to encode pyruvate dehydrogenase. Thus, there is no classical link between glycolysis and the tricarboxylic acid cycle. Interestingly, massively parallel signature sequencing has demonstrated that lactate dehydrogenase is the third most highly transcribed gene in T. parva schizonts [15], suggesting that in these organisms lactate may be the primary end product of glycolysis. This could be true for B. bovis as well. The enzymes adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) involved in salvage of purine bases appear to be lacking in B. bovis. HGPRT is present in P. falciparum (PF10_0121) [11], but absent from T. parva and T. annulata. Interestingly, although the purine salvage pathway is incomplete, B. bovis may be able to salvage purine nucleosides [16]. A recent analysis of B. bovis expressed sequence tags (ESTs) identified two adenosine kinases [17], a finding corroborated by the genome sequence data, which also revealed the presence of adenosine deaminase. These enzymes are absent in T. parva, while P. falciparum encodes adenosine deaminase. While we cannot exclude that HGPRT is present in the chromosome 1 gap, the apparent absence of HGPRT in B. bovis is in contrast to previous studies demonstrating the incorporation of radio-labeled hypoxanthine in parasite erythrocyte cultures [16,18]. Although several enzymes involved in purine salvage are present, there appears to be no direct path to the production of inosine monophosphate, and it is possible that the necessary enzymes are present but are not similar to known enzymes. Unlike P. falciparum and the Theileria spp., B. bovis does not appear to encode dihydrofolate synthase, which converts dihydropteroate to dihydrofolate. However, this deficiency could be compensated through importation via a folate/biopterin transporter (BBOV_IV002460) and increased dihydrofolate reductase–thymidylate synthase (DHFR-TS) activity. Consistent with a previous study using the Israel strain of B. bovis [19], the T2Bo DHFR-TS contains three of the four amino acid substitutions found in a mutant P. falciparum DHFR-TS with strong resistance to pyrimethamine, a DHFR inhibitor. An additional single point mutation is linked with the ability of B. bovis to develop strong resistance to pyrimethamine [19]. Babesia bovis has the smallest number of predicted membrane transporters [20] among the sequenced apicomplexan species (Table S1), but encodes more members of some families (for example, glucose-6-phosphate/phosphate and phosphate/phosphoenolpyruvate translocators, members of the drug/metabolite transporter superfamily). It encodes fewer members of the ABC efflux protein family than T. parva but has more transporters for inorganic cations, including a cation diffusion facilitator family protein that is absent in T. parva and other apicomplexans. Both B. bovis and T. parva lack aquaporins, the calcium:cation antiporters, and amino acid permeases that are present in the genome of P. falciparum. Orthologs of the different types of amino acid transporters cannot be identified in B. bovis, including the dicarboxylate/amino acid:cation (Na+ or H+) symporter family amino acid:cation symporter that is present in T. parva [10]. The Apicoplast Most members of the phylum Apicomplexa harbor a semi-autonomous plastid-like organelle termed the apicoplast, which was derived via a secondary endosymbiotic event [21]. The B. bovis apicoplast genome is 33 kbp and unidirectionally encodes 32 putative protein coding genes, a complete set of tRNA genes (25), and a small and large subunit rRNA gene (Figure S1). The B. bovis apicoplast genome displays similarities in size, gene content, and order to those of Eimeria tenella, P. falciparum, T. parva, and Toxoplasma gondii (Table S2; [22–24]). As observed with other apicoplast genomes, the B. bovis apicoplast genome is extremely A+T rich (78.2%), in contrast to the nuclear genome (58.2%). In addition to the apicoplast genome encoded proteins, it has been demonstrated in P. falciparum that proteins encoded by nuclear genes are imported into the apicoplast (reviewed in [25]) to carry out a variety of metabolic processes, including heme biosynthesis [26], fatty acid biosynthesis [27], and isoprenoid precursor synthesis via the methylerythrose phosphate pathway [28]. Nuclear encoded proteins targeted to the apicoplast of P. falciparum have a bipartite targeting sequence consisting of a signal peptide that directs the protein to the secretory pathway and an apicoplast transit peptide that redirects the protein from the default secretory pathway into the lumen of the apicoplast [29,30]. Analysis of the metabolic functions ascribed to the apicoplast in P. falciparum reveals that only the enzymes for isoprenoid biosynthesis are found in B. bovis. To detect additional apicoplast-targeted proteins, PlasmoAP, a program developed to predict apicoplast targeting for P. falciparum [31], was used and revealed only 14 additional candidate proteins. This result is, perhaps, not unexpected, as the program was trained with P. falciparum sequences and likely works well only for P. falciparum because of skewed codon usage resulting from the low G+C content of P. falciparum. A third approach included visual inspection of BLAST search outputs of the entire B. bovis proteome against the nr database (National Center for Biotechnology Information) for potential amino-terminal extensions. This search resulted in 25 potential apicoplast-targeted sequences that had non-apicomplexan homologs with significant E values and bona fide amino terminal extensions. In total, 47 proteins (the eight involved in the methylerythrose phosphate pathway, 14 SignalP sequences identified with PlasmoAP, and 25 proteins identified through BLAST and visual inspection for amino terminal extentions) are predicted to be targeted to the B. bovis apicoplast (Table S3), by far the fewest of any organism for which this type of analysis has been done. P. falciparum and T. parva are predicted to have 466 and 345 apicoplast-targeted proteins, respectively [10,32]. The paucity of proteins predicted to be targeted to the B. bovis apicoplast may partially reflect the biology of the organism, with fewer functions attributed to the B. bovis apicoplast compared to P. falciparum, but is more likely a reflection of the lack of appropriate prediction algorithms. The apicoplast has been an attractive target for development of parasiticidal drug therapies as the biosynthetic pathways represented therein are of cyanobacterial origin and differ substantially from corresponding pathways in the mammalian host [21,33]. A recent study of the apicomplexan T. gondii demonstrated that fatty acid synthesis in the apicoplast is necessary for apicoplast biogenesis and maintenance, and indicates that this pathway would be an ideal target for drug design [34]. Thus, the reduced metabolic potential of B. bovis has important ramifications for drug design, suggesting that drugs targeting fatty acid synthesis would not be effective against babesiosis due to the absence of this pathway. The Mitochondrion B. bovis contains a 6 kbp linear mitochondrial genome (Figure S2). It encodes three putative protein coding genes, including cytochrome c oxidase subunit I, III, and cytochrome b. These are membrane-bound proteins that form part of the enzyme complexes involved in the mitochondrial respiratory chain. Cytochrome b and c subunit III are encoded on the same strand, while cytochrome c subunit I is encoded on the opposite strand. This coding arrangement is identical to that of Theileria spp. but different from that of P. falciparum [7,11,35]. Each of the encoded proteins employs the universal ATG as the start codon, in contrast to the T. parva cytochrome c subunit I, which has an AGT start codon [35]. In addition to the three protein coding genes, the B. bovis mitochondrial genome includes at least five partial rRNA gene sequences ranging in size from 34 to 301 bp. All five rRNA sequences are homologous to parts of the large ribosomal subunit of rRNA. They are encoded on both strands of the mitochondrial genome with rRNA 1 and 5 on the same strand and 2, 3, and 4 on the opposite strand. A terminal inverted repeat was identified from position 11–180 and 6005–5836. Protein Families The B. bovis proteome was used to construct protein families using Tribe-MCL, a sequence similarity matrix-based Markov clustering method, and a method based on a combination of hidden Markov model domain composition and sequence similarity [36]. In addition to housekeeping gene families found in most eukaryotes, the pathogen contains only two large gene families. One of these families, encoding the variant erythrocyte surface antigen (VESA), has been previously defined [37]. The second, which we have termed SmORF (small open reading frame), is novel. Smaller notable families encode a 225 kD protein, known as spherical body protein 2 (SBP2) [38], and the variable merozoite surface antigen (VMSA) family [39]. VESA1. VESA1 is a large (>300 kD), heterodimeric protein composed of VESA1a and VESA1b that is synthesized by B. bovis and subsequently exported to the surface of the host erythrocyte [40]. VESA1 undergoes rapid antigenic variation and has been implicated in host immune evasion and cytoadhesion, both of which would be expected to play a vital role in persistence and pathogenesis [41,42]. VESA1 is thought to be the functional homolog of PfEMP1, encoded by the var gene family, in P. falciparum [43]. The ves1 genes comprise the largest family in the B. bovis genome. While sequence identity and the presence of similar secondary amino acid structures make it clear that these genes belong to the same family, two distinct types exist (ves1α and ves1β, encoding VESA1a and VESA1b, respectively) that possess highly variable regions of sequence composition, length, and gene architecture (Figure 3). Genomic analysis predicts 119 ves1 genes in the available sequence (72α, 43β, and four unclassified; Table S4). However, there is a gradient of increasing concentration of ves1 genes in the sequence immediately adjacent to the physical gap on chromosome 1, and the contigs that appear to reside in the gap contain ves1-like sequences, indicating that additional ves1 genes reside in the gap. An estimated gap size of 150 kbp would limit the number of genes within the missing sequence to less than 40, resulting in a total of approximately 150 ves1 genes, far fewer than previously predicted [37]. All but three members of the ves1 family are found in clusters of two or more genes, with individual clusters separated by a few kilobases to nearly one megabase. Interestingly, ves1 genes are distributed throughout all four chromosomes (Figure 1), in contrast to the observation that genes involved in antigenic variation, immune evasion, and sequestration, including P. falciparum var genes, are only occasionally found internally and are predominately telomerically located [11,44]. While ves1 genes are also found near telomeres and centromeres, 89 genes (75%) are located distal to these chromosomal structures. Figure 3 Diagram of a Locus Containing the ves1α, ves1β, and smorf Genes The genome backbone is a gray line, ves1α exons are blue, ves1β exons are red, and the SmORF exon is yellow. Introns are shown as blank boxes through which the genome backbone is seen. The systematic gene name for each gene is shown. Transmembrane helices (black bars), coiled-coil domains (green boxes), variant domains with conserved sequences 1 and 2 (pink boxes), and the SmORF signal peptide (orange box) are indicated. Arrows represent the positions of the primers for each of the cDNA experiments. The experiment number is indicated to the right of the primer sets, with experiment 1 targeting specific genes, experiment 2 sets of genes, and experiment 3 the published LAT. Transcription of ves1 genes has been hypothesized to occur at a “locus for active transcription” (LAT), described as a divergently oriented pairing of ves1α and ves1β genes [37]. This large locus encompasses nearly 13 kbp and includes ves1α and ves1β (each >4 kbp), a short intergenic region ( 91% sequence identity in pairwise comparisons while the ves1β cDNAs displayed a bimodal distribution, with 46% having >91% sequence identity and 50% having only 56%–70% sequence identity. The RNA used for these experiments was obtained from B. bovis T2Bo culture more than two years following isolation of the genomic DNA used to construct the libraries used for sequencing, possibly accounting for some of the sequence diversity, i.e., due to changes in the population represented in the culture at the time of sampling. However, although variation over time may account for some of the differences between the cDNA and genomic sequences, the number of unique sequences obtained from a single time point exceeds the number of predicted expression sites for ves1 genes. Consistent with this finding, numerous ves1 genes were also represented in EST data [17]. Var gene expression, while “leaky” in the ring stages of P. falciparum, appears to be restricted to a single, or very few, alleles in individual parasite populations in vivo [46,47]. However, multiple var transcripts, although far fewer than for B. bovis, have been detected when the organism is cultured in vitro [48]. One possible explanation for the large number of different ves1 cDNA transcripts may be that, similar to the var genes, ves1 genes are removed from in vivo transcriptional controls and/or phenotypic selection when the organism is grown in vitro. While in vivo analysis of ves1 transcription remains to be performed, the number of diverse transcripts is interesting, and may suggest more widespread transcription and alternative post-transcriptional control mechanisms than observed in other hemoprotozoa. SmORFs. Found associated with the ves1 genes across all four chromosomes are members of the second largest protein family (SmORFs) in the B. bovis genome (Figures 1 and 2). These “small open reading frames” (so named due to their association with, but smaller size than the ves1 genes) include 44 genes with lengths ranging from 381 to 1,377 nucleotides with no significant sequence identity to any protein or gene sequence in available databases. When compared to VESA proteins, a higher degree of sequence conservation (∼50% amino acid identity for most pairwise comparisons, with a range from 28%–95%) is found among SmORF paralogs, and 42/44 members consist of a single exon. Additionally, 43 family members are predicted to have a signal peptide, and all 44 are predicted by TMHMM [49] to exist extracellularly. Alignment of the SmORF sequences reveals four blocks of conserved sequence interrupted by linking sequence present in only one or a few of the SmORF paralogs (Figure S4). These long linking sequences interspersed between the blocks of identity in a few proteins account for the increased peptide length for the longer members of the family. Results from experiments designed to detect smorf transcripts were similar to that for ves1: two of five cloned products matched the predicted genome sequence while the remaining clones differed. The prevalence of canonical signal peptides among SmORFs, and their uniform association with ves1 clusters, tempts speculation that these proteins may play a functional role in VESA1 biology, or may, themselves, contribute to antigenic variation and immune evasion, or both. However, elucidation of the function of these proteins awaits biochemical and immunological analysis. SBP2 family. The spherical body is an apical organelle thought to be analogous to dense granules in other apicomplexan organisms [50]. SBP2 (also known as BvVa1) is a 225 kD immunostimulatory protein from the spherical body that is released into the host erythrocyte upon invasion and localizes to the cytoplasmic side of the erythrocyte membrane [38,51–53]. The original study noted that there were multiple copies of the 5′ end of the gene, while the 3′ end appeared to be single copy [51]. Consistent with this study, the genome sequence reveals that there are 12 truncated copies of the SBP2 gene corresponding to the 5′ end of the full-length gene, and one full-length copy. The full-length gene and one truncated gene are on chromosome 4, with all remaining truncated copies on chromosome 3. The truncated genes on chromosome 3 occur in three clusters of two, four, and five genes. The genes occurring in the 2- and 5-gene clusters are interspersed with another set of highly conserved (88%–100%) gene repeats (BBOV_III005620, BBOV_III006470, BBOV_III006490, BBOV_III006510, and BBOV_III006530) that have no homologs in the public databases. The 12 truncated SBP2 genes have sequence identities ranging from 27%–99% in pairwise comparisons, with the greatest identity in the first 30 amino acids. Previous analysis of EST sequences indicates that more than one of these truncated genes are transcribed [17]. VMSA. The variable merozoite surface antigen genes encode a family of immunostimulatory proteins that are a target of invasion blocking antibodies [39,54,55]. As in related Mexico strains of B. bovis [56], the T2Bo genome contains five vmsa genes, including msa1 and four copies of msa2. The VMSA genes reside on chromosome 1, with the four msa2 copies arranged tandemly in a head-to-tail fashion, and msa1 residing ∼5 kbp upstream from the msa2 genes. Interestingly, the VMSAs do not have homologs in Theileria spp. or P. falciparum. Comparative Analyses of Hemoprotozoan Proteomes Clusters of orthologous groups. Similarity clustering using the predicted proteomes of B. bovis, T. parva, and P. falciparum created 1,945 three-way clusters of orthologous groups (COGs) (Figure S5; Table S5). As expected from phylogenetic studies, the B. bovis proteome is more closely related to that of T. parva. Approximately half of the remaining B. bovis proteins not included in the three-way COGs fell into two-way COGs with proteins from T. parva, while B. bovis and P. falciparum shared only 111 two-way COGs. Remaining after cluster analysis were 706, 1,107, and 3,309 unique genes for B. bovis, T. parva, and P. falciparum, respectively (Tables S6–S8). Since P. falciparum, T. parva, and B. bovis all have complex life cycles that involve arthropod vector and mammalian host stages, Jaccard-filtered COG (Jf-COG) data were used to search for B. bovis orthologs of proteins that have been characterized in T. parva and P. falciparum as targets of protective immune responses, as well as those that play a role in stage-specific parasite biology. Many genes exclusively expressed in sexual stages of P. falciparum (for example PfCDPK3, PfLAMMER, and Pfs230) do not share Jf-COGs with T. parva or B. bovis, a difference potentially associated with the different vector (mosquito versus tick) that transmits Plasmodium. Likewise, P. falciparum sporozoite genes that are expressed initially in the mosquito, such as Pfs 25/28, exported protein 2, circumsporozoite protein, circumsporozoite protein/thrombospondin-related anonymous protein-related protein, and sporozoite microneme protein essential for cell traversal 1 and 2, also do not cluster with T. parva or B. bovis proteins. Since B. bovis does not have a pre-erythrocytic liver stage, as expected, orthologs of P. falciparum liver stage proteins such as PfLSA 1–3 are not detected. P. falciparum erythrocytic stage proteins such as the PfMSPs were not detected in B. bovis, nor were plasmodial rhoptry and rhoptry-associated proteins (RAPs). However, BbRAP-1a (BBOV_IV009860 and BBOV_IV009870) forms a Jf-COG with its T. parva (TP01_0701) and T. annulata (TA05760) orthologs. Interestingly, B. bovis encodes a protein (RRA; BBOV_IV010280) most similar to RAP-1b, previously described only in B. bigemina [57]. Noteworthy P. falciparum genes that have Jf-COGs with B. bovis are thrombospondin-related anonymous protein (TRAP), p36 protein, Pf12, Sir2, PfATP6, and P0. PfTRAP is expressed exclusively in sporozoites, while BbTRAP is expressed in both sporozoite and blood stages [58]. A plasmodial surface membrane protein, p36 is a member of the p45/48 sporozoite protein family. It participates in liver stage parasite development, and immunization with Pfp36 knockout parasites results in protective immunity against subsequent challenge with wild-type sporozoites, identifying p36 as a potential knockout gene for development of attenuated vaccines [59]. Pf12 is a merozoite surface protein that is recognized strongly by antibodies of naturally infected individuals [60]. An ortholog of the Sir2 protein, involved in P. falciparum var gene silencing [61], is present in B. bovis (BBOV_I003070), forming a Jf-COG with orthologs in P. falciparum (PF13_0152), T. parva (TP01_0527), and Cryptosporidium parvum (cgd7_2030), but is apparently absent from T. annulata. An ortholog of PfATP6, the gene thought to be the target of the drug artemisinin used to treat drug-resistant malaria, is found in B. bovis (BBOV_II005700) [62]. Finally, BBOV_IV004540 forms a Jf-COG with P0 from P. falciparum (PF11_0313), T. parva (TP01_0294), and T. annulata (TA21355). P0 is a ribosomal phosphoprotein with immunoprotective properties [63]. Immunostimulatory proteins that form Jf-COGs with B. bovis include a T. parva protein annotated as polymorphic immunodominant protein (PIM) (TP04_0051). This polymorphic immunodominant protein is the target of sporozoite neutralizing antibodies [64], and falls into a Jf-COG with B. bovis protein BBOV_II005100, T. annulata protein TA17315, known as TaSP [65], and P. falciparum protein PF14_0369. However, the orthologs are half the length of the T. parva protein and do not contain a Q/E-rich central repeat domain that is characteristic of PIM. Of six additional antigens from T. parva (TP01_0056, TP02_0849, TP02_0767, TP02_0244, TP02_0140, TP03_0210) that are the targets of parasite-specific bovine MHC class I–restricted CD8+ cytotoxic T cells [66], four have orthologs in B. bovis (BBOV_IV000410, BBOV_IV006970, BBOV_III011550, BBOV_III004230, BBOV_III010070) and P. falciparum (PFC0350c, PF13_0125, MAL7P1.14, PF11_0447, PF14_0417, PF07_0029). BBOV_IV000410, one of the genes not found in P. falciparum, encodes a signal peptide-containing protein whose T. annulata homolog is targeted to the membrane [67]. B. bovis ACS-1 (BBOV_III010400) has been shown to stimulate CD4+ T lymphocyte responses in immune cattle [68], and forms a Jf-COG with T. parva (TP02_0107) and P. falciparum (PFL1880w) proteins. The B. bovis apical membrane antigen 1 (AMA-1; BBOV_IV011230) [69] is a micronemal protein that forms a Jf-COG with P. falciparum (PF11_0344), T. parva (TP01_0650), and T. annulata (TA02980) and has additional homologs with other apicomplexans. The B. bovis AMA-1 gene is located on chromosome 4 and is part of a syntenic cluster of four genes present across the P. falciparum, T. parva, and T. annulata genomes. A unique aspect of T. parva and T. annulata is the ability of the schizont stage of these parasites to transform the leukocytes they reside in to a cancer-like phenotype [70]. This reversible change is dependent on the presence of viable parasites. Although a number of Theileria molecules that could interfere with host cell signaling pathways controlling cell proliferation and apoptosis have been mined from the genome sequence of both pathogens, no single molecule in either parasite could be linked with the phenotype. In general, both parasites encode the same repertoire of candidate proteins, suggesting that subtle differences account for the observation that T. parva transforms T and B cells while T. annulata transforms B cells and macrophages. As anticipated, the expansion in the number of genes coding for choline kinase in T. parva and T. annulata, which may contribute to increased lipid metabolism in transformed cells, is not present in B. bovis, which encodes a single copy of this gene. In an effort to further refine a list of candidate transformation-associated genes for T. parva, we analyzed a list of 1,107 T. parva proteins that do not fall into a Jf-COG with proteins from P. falciparum or B. bovis (Table S7). There are 262 proteins predicted to contain a signal peptide or signal anchor and are not predicted to be targeted to the apicoplast. Cross-referencing this list with transcriptional data derived from oligonucleotide based microarrays comparing T. parva schizonts and sporozoites reveals that 35 genes in the list are highly expressed in schizonts. These include two members of the TashAT gene family previously implicated in T. annulata transformation [71], and one member of a telomeric gene family [7]. It is notable that the remaining genes are all annotated as hypothetical proteins, emphasizing the need for a concerted effort to study the role of these novel proteins. Syntenic analyses. It is possible that due to evolutionary pressure, functional B. bovis homologs of T. parva and P. falciparum proteins may have diverged in sequence to the point they are no longer recognizable at the level of the primary amino acid sequence. For this reason, we examined the conservation of gene order in syntenic blocks between the pathogens. Syntenic blocks were defined as a pair of genes that belong to the same Jf-COG, where members of the pair belong to the reference and query sequence [10]. Even by this method, we were unable to identify obvious homologs for many P. falciparum proteins involved in stage-specific biology or host immunity. However, in T. parva, the regions flanking a gene encoding an abundant sporozoite surface antigen, p67, a primary target of parasite neutralizing antibodies [72], form a highly conserved syntenic block with B. bovis and T. annulata (Figure S6). Sporozoite antigen 1 (SPAG-1), the positional homolog of p67 in T. annulata, is itself known to contain neutralizing epitopes and is a leading vaccine candidate [73]. The gene in B. bovis (BBOV_IV007750) that occupies the site of p67 in T. parva is predicted to encode a membrane protein, suggesting that this protein may have immunostimulatory properties equivalent to p67. RT-PCR experiments indicate that the B. bovis gene is transcribed in infected erythrocytes and during the kinete stage in ticks (unpublished data; sporozoite expression has not been examined). It will be interesting to explore the vaccine potential of the B. bovis p67 homolog, as ∼50% of cattle immunized with recombinant p67 and challenged under field conditions show a reduction in severe East Coast fever [74]. Large blocks of synteny are evident between B. bovis and T. parva chromosomes (Figure 4A). However, several chromosomal rearrangements have taken place, as observed between chromosomes of P. falciparum and P. yoelii yoelii [75]. Synteny rarely extends to telomeres (Figure 4B), as these regions usually contain species-specific polymorphic genes that are present at many syntenic break points. Unlike the T. parva subtelomeric regions, the B. bovis subtelomeres contain genes transcribed from both strands. However, similar to both T. parva and P. falciparum, the telomeres contain many (putative) membrane proteins. At a gross level, B. bovis chromosomes 2 and 4 primarily consist of sections of T. parva chromosome 4 and 2, and 3 and 1, respectively. B. bovis chromosome 3 contains sections from all four T. parva chromosomes, while B. bovis chromosome 1 contains DNA from T. parva chromosomes 3, 1, and 2 (Figure 4). Closer examination of syntenic blocks indicates that inversions in gene order have also taken place. Figure 4 Diagram of Chromosomal Synteny between B. bovis and T. parva (A) Synteny at the chromosomal level. B. bovis chromosome number is indicated at the top. Bars on the right side of each chromosome diagram designate B. bovis genes, with black bars indicating B. bovis ves1 genes and gray bars indicating other genes. The colors on the left of each chromosome diagram indicate to which T. parva chromosome an ortholog belongs as follows: Tp1 = red, Tp2 = green, Tp3 = blue, Tp4 = orange. (B) Comparison of telomeric arrangement of genes for B. bovis and T. parva chromosome 2. The gray line indicates the chromosomal backbone, with black dots indicating the telomere. Large genes are depicted as arrows with coding direction indicated, while small genes have an arrowhead beneath the gene to indicate the direction of transcription. Gray double arrowheads indicate that the chromosome continues. Colors indicate gene content as follows: blue = B. bovis ves1α, red = B. bovis ves1β, yellow = B. bovis SmORF, pale green = putative membrane protein, pale blue = annotated genes with predicted function, black = hypothetical, orange = T. parva family 3 hypothetical, purple = T. parva family 1 hypothetical, green = ABC transporter. Summary The 8.2 Mbp genome of B. bovis consists of four nuclear chromosomes, and two small extra-nuclear chromosomes for the apicoplast and mitochondria. B. bovis appears to have one of the smallest apicomplexan genomes sequenced to date. Consistent with the small genome size, analysis of enzyme pathways reveals a reduced metabolic potential, and provides a better understanding of B. bovis metabolism and potential avenues for drug therapies. Using several different approaches, identification of proteins predicted to be targeted to the apicoplast reveals far fewer proteins than for related organisms. This may be due in part to the lack of appropriate detection algorithms. However, the conservative approach used to identify the genes encoding these proteins provides a solid base from which to extend these analyses. A foundation for the elucidation of antigenic variation and immune evasion has been established with genome-wide characterization of the ves1 gene family, and discovery of the novel smorf gene family. ves1 and smorf genes are co-distributed throughout the chromosome, with the majority located away from telomeres and centromeres. As many as 33 potential loci of ves1 transcription have been identified, and cDNA analysis suggests that this transcription is more broad-based than with other hemoprotozoa. Comparative analysis indicates that many stage-specific and immunologically important genes from P. falciparum are absent in B. bovis. However, through both COG analysis and synteny, additional B. bovis vaccine candidates, including homologs of P. falciparum p36, Pf12, T. parva p67, and four of six T. parva proteins targeted by CD8+ cytotoxic T cells, have been identified. Methods Parasite culture and library construction. R. microplus adults were allowed to feed on calf C-912 inoculated with the T2Bo strain that was one passage (splenectomized calf) removed from a field isolate and frozen as a liquid nitrogen stabilate [76]. Progeny larvae were placed on calf C-936, blood was collected 7 d post tick infestation, and microaerophilous stationary phase culture was established according to [77] with modifications as described in [18]. Parasite genomic DNA from parasites in culture for 34–39 weeks was extracted using standard methods [78]. Small (2–3 kbp) and medium (12–15 kbp) insert libraries were constructed by nebulization and cloning into pHOS2. A large insert library (100–145 kbp) was constructed in pECBAC1 (Amplicon Express) and consisted of clones resulting from HindIII or MboI partially digested DNA. Genome sequencing. A total of 103,478 high quality sequence reads (average read length = 870) were generated (58,251 reads from the small insert library and 45,227 reads from the medium insert library) and assembled using Celera Assembler (http://sourceforge.net/projects/wgs-assembler/). The sequence data fell into 50 scaffolds consisting of 88 contigs. The bacterial artificial chromosome library was end sequenced to generate an additional 2,874 reads that were used to confirm the assembly and for targeted sequencing in the closure phase. Gaps in the assembly were closed by a combination of primer walking and transposon based or shotgun sequencing of medium insert clones, bacterial artificial chromosome clones, or PCR products. This genome project has been deposited at DDBJ/EMBL/GenBank under accession number AAXT00000000. The version described in this paper is the first version, AAXT01000000. Functional annotation. Chromosomal gene models were predicted using Phat [79], GlimmerHMM, TigrScan [80], and Unveil [81] after training each gene finding algorithm on 499 partial and full-length B. bovis genes totaling ∼453 kbp. The training data were manually constructed after inspection of the alignment of highly conserved protein sequences from nraa using the AAT package [82] and PASA to align a collection of ∼11,000 B. bovis ESTs [17] to the genome sequence. Jigsaw was used to derive consensus gene models [83] from the outputs of the gene finding programs and protein alignments. The consensus gene models were visually inspected and obvious errors such as split or chimeric gene models were corrected based on either EST or protein alignment evidence using the Neomorphic Annotation Station [84] before promotion to working gene models. Genes encoding tRNAs were identified using tRNAscan-SE [85]. BLAST [86] was used to search nraa using the predicted B. bovis protein sequences, and protein domains were assigned using the InterPro database [87]. The presence of secretory signals and transmembrane domains were detected using SignalP [88] and TMHMM [49], respectively. Functional gene assignments were assigned based on the BLAST data, and a Web-based tool called Manatee (http://manatee.sourceforge.net/) was used to manually curate and annotate the data. Proteins were annotated as hypothetical proteins if there was less than 35% sequence identity to known proteins, and as conserved hypothetical proteins if there was greater than 35% sequence identity to other proteins in the database that were unnamed. If a protein was predicted to have a signal peptide and at least one transmembrane domain, but was otherwise considered as a hypothetical or conserved hypothetical protein, it was annotated as a membrane protein, putative. If there was greater than 35% sequence identity for 70% of the sequence length, the protein product would be assigned a name only when a publication record could verify the authenticity of the named product. In the absence of published evidence, the named product was listed as putative. The mitochondrial and apicoplast genomes were manually annotated, and apicoplast-targeted proteins were analyzed using PlasmoAP (http://v4-4.plasmodb.org/restricted/PlasmoAPcgi.shtml) [31]. PASA [89] was used to align ∼86% of the B. bovis ESTs to the genome sequence data and provided evidence for transcription of 1,633 genes. Comparative analyses. Sybil (http://sybil.sourceforge.net/) was used to create an all-versus-all BLASTP search using the proteomes of B. bovis, T. parva, and P. falciparum. These outputs were subjected to Jaccard clustering [10], placing proteins into distinct clusters for each proteome. Clusters from different proteomes were linked based on best bidirectional BLASTP hits between them to provide Jf-COGs. A minimum block size of five with one gap was allowed in the analyses. cDNA analyses. Analysis of ves1 transcription utilized total RNA isolated from microaerophilous stationary phase culture culture using TRIzol (BRL) treated three times with RNase-free DNase (Ambion) for 30 min at 37 °C. RNA was reverse transcribed with a Superscript (Invitrogen) reverse transcription kit using random hexamers according to the manufacturer's instructions. Universal primer sequences that would anneal to the two specific subunit types could not be found. Therefore, in the first RT-PCR experiment, primers were designed to amplify as many of the genes as possible. The following primers were used for ves1β cDNA: beta2For: 5′ GGA CTA CAG AAG TGG GTT GGG TGG and beta4Rev: 5′ ATA GCC CAT GGC CGC CAT GAA TGA; ves1α cDNA: alpha3For: 5′ CAG GTA CTC AGT GCA CTC GTT GGG TGG AG and alpha6Rev: 5′ CCC TAA TGT AGT GNA CCA CCT GGT TGT ATG C. Due to the high degree of sequence similarity (>99%) of the published ves1 loci in cosmid 53 and 54 (accession numbers AY279553 and AY279554, respectively) to the genome sequence, a second RT-PCR experiment used primers designed to amplify the published LAT [37]. This experiment used primers LATbetaF1: 5′ GCA ACC GCA CGA CAG and LATbetaR2: 5′ CGC TGA CAC GCT AGT for the ves1β gene. A final cDNA cloning experiment was designed to elucidate the transcriptional profile for ves1 by targeting ves1α and ves1β genes associated with Rep sequence clusters [37]. Primers were as follows: ves1β: 00789F1: 5′ AGA CTG TGA ATC TCG GCT CA and 00789R: 5′ CAG CGG CAC CAC TAC CTT T; ves1α: 00792F2: 5′ TGC CCA GGA CAG TTA TG and 00792R2: 5′ TGA TGC CCT CTT CAA TAG TT. Whenever possible, ves1 primers were designed such that they would flank introns, providing an additional assurance that the amplicon obtained was not from contaminating genomic DNA; however, this was only possible for ves1β experiments. Supporting Information Figure S1 Diagram of B. bovis Apicoplast Genome Coding sequences (CDSs) with functional annotation are depicted in red, while hypothetical CDSs are shown in lack. Genes encoding tRNA genes are shown as blue bars, while rRNA genes are shown as yellow arrows. All genes are unidirectionally encoded. (303 KB PDF) Click here for additional data file. Figure S2 Diagram of B. bovis Mitochondrial Genome CDSs are depicted in red, large subunit rRNA genes in blue, and inverted terminal repeats as black arrows. (38 KB PDF) Click here for additional data file. Figure S3 Diagram of the 24 Putative ves1 LAT Loci All loci are depicted with ves1β on the left and ves1α on the right and drawn to the same scale. The genome backbone is a yellow line, exons are orange, and introns are shown as blank boxes. The systematic gene name for each gene is shown. Transmembrane helices (TMHelix), coiled-coil domains (Coiled Coil), and the variant domains with conserved sequences 1 and 2 (VDCS-1 or −2) are indicated. (327 KB PDF) Click here for additional data file. Figure S4 Alignment of 44 SmORF Sequences Deduced amino acid sequences were aligned with the AlignX module of VectoNTI. The blue background indicates positions with identical amino acid sequences, while the green background indicates conserved amino acids. Dashes indicate that there is no amino acid in that position. Long stretches of intervening amino acid sequence has been trimmed from a few sequences to allow visualization of the four blocks of amino acid conservation. The double slashes (//) indicate that the sequence was trimmed from this position that spanned the full length between the remaining amino acids, while a single slash (/) indicates that the sequence that was trimmed from the alignment that did not fully span the region was removed. The total alignment length is 720 amino acids in length. (43 KB PDF) Click here for additional data file. Figure S5 Venn Diagram Showing Number of Genes in Overlapping COGs between B. bovis, T. parva, and P. falciparum (14 KB PDF) Click here for additional data file. Figure S6 Example of Microsynteny between B. bovis and T. parva The T. parva p67 locus is diagrammed in the middle row, with the corresponding B. bovis locus shown on the top row, and the T. annulata SPAG-1 locus diagrammed on the bottom row. Genes with sequence identity between species are connected by shaded gray lines. The gene highlighted with pink was used to identify the syntenic locus. The p67 gene is indicated. (52 KB PDF) Click here for additional data file. Table S1 Transporters (85 KB DOC) Click here for additional data file. Table S2 Characteristics of Apicomplexan Plastids (34 KB DOC) Click here for additional data file. Table S3 Nuclear Encoded Genes Potentially Targeted to the Apicoplast (104 KB DOC) Click here for additional data file. Table S4 Characteristics of ves1 Sequences (44 KB XLS) Click here for additional data file. Table S5 CDS common to B. bovis, T. parva, and P. falciparum (728 KB XLS) Click here for additional data file. Table S6 CDS Unique to B. bovis (97 KB XLS) Click here for additional data file. Table S7 CDS Unique to T. parva (138 KB XLS) Click here for additional data file. Table S8 CDS Unique to P. falciparum (388 KB XLS) Click here for additional data file. Accession Number The B. bovis T2Bo genome is deposited at DDBJ/EMBL/GenBank under accession number AAXT00000000.

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Intracellular parasite invasion strategies.

L. Sibley (2004)

Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called "gliding" to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

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Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion

Daniel Bargieri, Nicole Andenmatten, Vanessa Lagal … (2013)

Most apicomplexans, including the agents of malaria (Plasmodium) and toxoplasmosis (Toxoplasma), are obligate intracellular parasites. Many invade host cells by a conserved mechanism involving the formation of a zone of tight attachment between the parasite apex and the host cell1, called tight junction (TJ). The current view is that the TJ is primarily a molecular bridge between the parasite sub-membrane actin-myosin motor and a stable and stationary2 anchor associated with the host cell surface/cortex, which allows parasite traction into a parasitophorous vacuole (PV) within the host cell3 4. The transmembrane protein apical membrane antigen 1 (AMA1), arguably the most studied protein in the Apicomplexa phylum and a long-standing malaria vaccine candidate, is thought to shape the TJ on the parasite side5 6 7 8 9 10 11. The cytoplasmic tail of AMA1 was reported to bind aldolase8 in vitro, considered a signature of proteins that bind parasite actin and the motor12 13. The ectodomain of AMA1 tightly binds in parasite extracts to the rhoptry neck 2 (RON2) protein9 10, a protein secreted from the parasite rhoptries that specifically localizes at the TJ, where it inserts in the host cell membrane and is presumably linked to the cell cortical cytoskeleton via other RON proteins, like RON4. Moreover, antibodies or peptides that inhibit the AMA1–RON2 interaction drastically reduce host cell invasion by Plasmodium merozoites14 15 16 and Toxoplasma tachyzoites9 10. T. gondii or P. falciparum AMA1 bound to RON2 peptide were co-crystalized, revealing a conserved RON2 loop inserting deep into an AMA1 hydrophobic groove17 18. This reinforced the model of this interaction constituting the traction point used by the parasite to power the active internalization inside the PV19 20 21, and led to the proposal of developing broad-spectrum small-molecule inhibitors of apicomplexan invasion targeting the AMA1–RON2 interaction22 23 24. In addition, AMA1 has been reported to be involved in rhoptry secretion15 25 as well as for providing a signal initiating intracellular replication26. Recently, P. berghei and T. gondii parasites in which AMA1 was silenced (AMA1 knockdown, AMA1KD) were found to remain competent for shaping a TJ and invading host cells27. However, as AMA1KD parasites might still express residual levels of AMA1, these data were not considered as challenging any of the proposed roles of AMA1 in invasion18 23 28 29. In agreement with an essential role of AMA1 at some stage of the parasite invasion process, all attempts to inactivate AMA1 in both Plasmodium 30 31 and Toxoplasma 32 have failed so far. Here, we report the inactivation of AMA1 in the tachyzoite of T. gondii, which invades virtually any cell type in the host, and in the merozoite and sporozoite stages of P. berghei, which invade erythrocytes and hepatocytes, respectively. AMA1 was deleted from the parasite genome by the diCre-loxP recombination approach in T. gondii and by direct homologous recombination in P. berghei. All three AMA1 knockout (AMA1KO) zoites are still capable of penetrating the respective host cell like the wild type (WT). Tachyzoites and merozoites, however, display a defect in host cell binding. These genetic data indicate that AMA1 and the RON proteins act separately during apicomplexan invasion, and that the AMA1–RON2 interaction does not have an essential role at the TJ. Results Role of AMA1 in T. gondii tachyzoite infection of host cells To investigate the role of AMA1 in T. gondii tachyzoites, we generated AMA1KO parasites using the diCre-loxP site-specific recombination system33. The loxPAMA1loxP-YFP-HXGPRT construct (Fig. 1a) was inserted in the ku80::diCre strain, which encodes two inactive fragments of Cre fused to rapamycin-binding proteins. Upon rapamycin addition and Cre reconstitution, recombinant parasites excise AMA1 (Fig. 1b) and express YFP. An excised clone, called TgAMA1KO, which does not produce AMA1 as shown by western blot (Fig. 1c) and immunofluorescence (Fig. 2a) analysis, was selected and maintained using routine culture procedures. When measuring parasite infectivity by plaque assay, the plaque size is ~2 to 2.5 times smaller with TgAMA1KO than control parasites (Fig. 2a,b), a rather mild phenotype. Reintroduction of internally tagged AMA1FLAG in TgAMA1KO parasites fully restores overall growth, demonstrating the specificity of the observed phenotype (Fig. 2a). Importantly, no significant difference can be noticed in the replicative rates (Fig. 2c,d), egress efficiency (Fig. 2e) or motility patterns (Fig. 2f) between TgAMA1KO and control tachyzoites. Therefore, AMA1 is not involved in tachyzoite gliding, intravacuolar replication or egress from host cells. We then investigated AMA1KO tachyzoite invasion of host cells in more detail. When measured by fluorescence microscopy, TgAMA1KO tachyzoite invasion efficiency is 30–40% that of the parental ku80::diCre strain (Fig. 3a). To investigate whether the pattern of TgAMA1KO tachyzoite invasion of host cells is normal or altered, TgAMA1KO tachyzoites invading human foreskin fibroblasts (HFFs) or normal rat kidney (NRK) fibroblasts were captured by confocal microscopy and analysed after three-dimensional reconstruction (Fig. 3b). Entering mutant zoites (n=53) constantly display a typical RON4 circular staining around the parasite constriction site after cell permeabilization, indicating normal rhoptry secretion and TJ formation during invasion. In line with the normal gliding capacity of TgAMA1KO tachyzoites, micronemal protein 2 (MIC2)34, which is secreted like AMA1 from the microneme organelles, is normally exposed on the surface of invading TgAMA1KO tachyzoites (Fig. 3b). Moreover, video microscopy of invading TgAMA1KO zoites shows that successful invasion follows a one go and smooth process with similar kinetics as controls (Fig. 3c,d and Supplementary Movies 1–3), and in all cases (n=20) a clear constriction site, suggestive of normal TJ, is observed. We conclude that in the tachyzoite AMA1 is not necessary for structuring a fully functional TJ, in which the RON proteins act independently of AMA1. Next, we assessed tachyzoite adhesion to host cells. After 15 or 60 min incubation with live HFF cell monolayers, approximately two- to threefold fewer mutant versus control tachyzoites associate with host cells (total: extracellular attached and internalized combined), whereas three- to fourfold fewer mutants are located inside host cells, suggesting a primary defect of mutant zoites in host cell attachment (Fig. 3a). Importantly, about a third of the TgAMA1KO tachyzoite population adopts a distinct position relative to the host cell than controls, by binding only via the apical end rather than throughout their length (Fig. 3e), like previously observed for AMA1KD tachyzoites27. This confirms that AMA1 has an important role in tachyzoite adhesion to/positioning onto host cells before TJ formation, an event that favours, but is not required for, host cell invasion. Role of AMA1 in P. berghei merozoite infection of erythrocytes To inactivate AMA1 in P. berghei, WT ANKA blood stages were transfected with a construct designed to replace endogenous AMA1 by pyrimethamine-resistance and green-fluorescence cassettes (Fig. 4a). Dedicated in vivo selection protocols with several days of drug pressure reproducibly generated mixtures of targeted green fluorescent protein (GFP+) AMA1− parasites, that is, AMA1KO, and non-targeted GFP− AMA1+ parasites, presumably spontaneous pyrimethamine-resistant mutants that typically emerge after long selection times. Southern blot analysis indicates the presence in the selected population of both the WT AMA1 locus and the expected allelic replacement (Fig. 4b). In agreement with this, immunofluorescence assays reveal erythrocytes infected by either GFP+ AMA1− or GFP− AMA1+ parasites (Fig. 4c). The multiplication rate of AMA1KO parasites, assessed by co-injection with control red fluorescent protein (RFP+) parasites35 in mice and monitoring parasite multiplication by fluorescence-activated cell sorting (FACS), is ~35% that of RFP+ parasites (Fig. 4d). As internalized AMA1KO parasites generate normal numbers of progeny merozoites after a normal developmental cycle (Fig. 4e), that is, AMA1 is not important for merozoite replication inside erythrocytes, the decreased multiplication rate of AMA1KO parasites reflects a defect in merozoite entry into erythrocytes. We next characterized interactions between AMA1KO merozoites and erythrocytes using imaging flow cytometry (IFC), which combines microscopy and flow cytometry and provides quantitative and functional information using imaging algorithms. Briefly (see Methods), after mixing mouse erythrocytes pre-stained with the lipid dye PKH26 with P. berghei GFP+ merozoites36 collected from synchronized schizont cultures, parasites interacting with a host cell are identified as GFP signals in a gated PKH26+ population (Fig. 5a, left panel), and internalized parasites are further recognized by co-localization of GFP with an increased PKH26 signal relative to the rest of the cell (Fig. 5b), a labelling suggestive of merozoites surrounded by a tight-fitting vacuole membrane37 (Fig. 5a, right panel). Using control GFP+ merozoites incubated for 10 min with PKH26-stained erythrocytes before fixation, ~43.8% score as ‘associated’ with erythrocytes (EryA; Fig. 5a, left panel), whereas ~3.8% score as ‘internalized’ inside erythrocytes (EryI; Fig. 5a, right panel). Importantly, cytochalasin D, which prevents merozoite internalization but not attachment to erythrocytes38, does not significantly affect the EryA but drastically reduces the EryI population (Fig. 5a,c), which validates the EryI population algorithm. Using merozoites of the AMA1KO-containing population, ~8.6% of the GFP+ AMA1KO merozoites score as EryA and ~0.48% as EryI (Fig. 5d). A similar reduction relative to control merozoites is obtained when samples are fixed after 3 min incubation (Fig. 5e), indicating a primary defect in adhesion of AMA1KO merozoites. Like AMA1KO tachyzoites, AMA1KO merozoites form a normal RON (RON2) ring during host cell invasion (Fig. 4f). As additional mutations, compensatory or adverse, might accumulate in the AMA1KO parasites propagated for extended times (up to 30 days) before IFC analysis, we next characterized AMA1KD merozoites generated by Flippase (Flp)/Flp Recombination Target (FRT)-mediated recombination27 immediately before IFC (Fig. 6a). In this approach, AMA1KD mosquito-stage sporozoites normally invade hepatocytes and transform into AMA1KD hepatic merozoites27. The latter cannot accumulate compensatory mutations before IFC, as they are generated in the absence of selection pressure and following a single invasion/multiplication cycle. We first analysed control hepatic merozoites. IFC analysis shows that ~44.3% and ~4.1% of control GFP+ hepatic merozoites score as EryA and EryI, respectively, indicating that erythrocytic and hepatic merozoites bind and invade erythrocytes with similar efficiency in this assay. We then used AMA1KD hepatic merozoites, composed of ~85% of excised AMA1− parasites lacking any detectable AMA1 and ~15% of non-excised AMA1+ individuals used as internal controls (Fig. 6b). IFC analysis after AMA1 immunostaining (Fig. 7a) shows that ~48.8% and ~5.3% of AMA1+ controls score as EryA and EryI, respectively, indicating that they behave like the WT (Fig. 7b). In contrast, only ~3.3% of AMA1− merozoites score as EryA (Fig. 7b), that is, ~15-fold less than internal controls, demonstrating a major role of AMA1 in merozoite attachment. As expected, AMA1− merozoites also generate EryI events after 10 min (Figs. 6c and 7b) or 3 min incubation (Fig. 7c). Remarkably, EryI events are approximately fivefold less frequent in AMA1− than AMA1+ merozoites when normalized to input merozoites, but approximately threefold more frequent in AMA1− merozoites when normalized to attached parasites (Fig. 7b, P<0.01, two-tailed t-test). Therefore, as with the Toxoplasma tachyzoite, AMA1 favours Plasmodium merozoite attachment to, but not internalization into, the host cell. AMA1 has no role in P. berghei sporozoite infection of hepatocytes Recent work using P. berghei AMA1KD and RON4KD sporozoites revealed strikingly distinct phenotypes, with essential and dispensable roles for RON4 and AMA1, respectively, during sporozoite invasion of hepatocytes27. To test AMA1KO sporozoite capacity to invade hepatocytes, populations of GFP+ AMA1KO/GFP− AMA1+ parasites were transferred to mosquitoes. The same ratio of GFP+ versus GFP− sporozoites is found in the blood fed to mosquitoes and in the mosquito salivary glands, indicating that AMA1 has no detectable effect on parasite development in the mosquitoes (Fig. 8a). The capacity of these salivary gland sporozoites to invade cultured hepatocytes was then tested. After sporozoite incubation with HepG2 cells in vitro, a similar proportion of AMA1KO versus GFP− AMA1+ parasites is found in the input sporozoites and in hepatic schizonts developing inside HepG2 cells 60 h post infection (Fig. 8b). Likewise, in co-infection experiments of HepG2 cells with RFP+ AMA1+ as control, AMA1KO sporozoites display similar invasive capacity as the control (Fig. 8c). Finally, the infectivity of AMA1KO sporozoites was tested in vivo. We found that intravenous injection into mice of as few as 500 AMA1KO/AMA1+ sporozoites (Fig. 8d) or HepG2 cell-released hepatic merozoites (not shown) is sufficient to generate blood-stage parasite populations containing AMA1KO parasites, demonstrating that parasites can complete a life cycle without producing AMA1. Moreover, injection into mice of only 50 AMA1KO/AMA1+ infected erythrocytes is also sufficient to produce AMA1KO-containing blood-stage populations (not shown). However, attempts of cloning AMA1KO parasites were unsuccessful. This is likely due to the slower increase in parasitemia of AMA1KO parasites, delaying the emergence of an AMA1KO population that is eventually cleared by the mouse immune system before being detectable. Nonetheless, we cannot rule out the formal hypothesis that AMA1KO parasites cannot be cloned because they require soluble AMA1 secreted from the AMA1+ counterparts. However, this hypothesis of AMA1 as an essential diffusible factor appears unlikely, as AMA1KO growth is observed after co-injection of less than 50 blood stages and 500 sporozoites in the whole animal. Discussion We have inactivated AMA1 both in Toxoplasma and Plasmodium using diCre-loxP-mediated recombination and direct gene targeting, respectively, and found that AMA1-deficient T. gondii tachyzoites and P. berghei merozoites and sporozoites were still invasive and displayed a normal host cell penetration step. The most striking phenotype is that of AMA1KO sporozoites, which showed no defect in hepatocyte invasion, confirming prior data obtained with AMA1KD sporozoites that invaded hepatocytes even better than the WT27. This now demonstrates that AMA1 is dispensable for hepatocyte invasion and that, given the essential role of RON4 in the process27, the RON complex acts in an AMA1-independent manner. The lack of an invasion phenotype of AMA1KO sporozoites strongly suggests that AMA1 is not involved in TJ function. In contrast to sporozoites, AMA1-deficient merozoites and tachyzoites displayed an approximately three- to fivefold decrease in overall invasion efficiency. However, like sporozoites, they penetrated host cells like the WT. They formed a normal constriction and a normal RON ring at the TJ, and tachyzoites were internalized at the normal average speed of ~20 s. Moreover, quantitative IFC analysis indicated that AMA1KD merozoites invaded erythrocytes better than controls when normalized to adherent merozoites, reminiscent of the increased infectivity of AMA1KD sporozoites. The decrease in invasion efficiency of AMA1-deficient tachyzoites and merozoites, which showed no defect in host cell penetration, was associated with altered zoite adhesion to host cells. Fewer AMA1-deficient merozoites bound to erythrocytes in IFC experiments, including in 3′ adhesion assays. Lack of AMA1 only modestly reduced the numbers of bound tachyzoites but affected their positioning onto cells, with AMA1-deficient tachyzoites more frequently adopting an upward position when compared with controls. AMA1 might thus be important in a pre-invasive zoite orientation step, as earlier proposed for merozoites39. A gradient of AMA1 on the zoite surface might create a gradient of interaction forces in a Velcro-like mechanism that might either apically reorient a zoite-expressing AMA1 mostly at its front end (merozoite) or flatten a zoite-expressing AMA1 all over its surface (tachyzoite). Why AMA1 has zoite-dependent contributions is unclear but might be related to zoite shape. A zoite-specific optimal positioning step, possibly involved in inducing rhoptry secretion, might be useful for the pear-shaped tachyzoites and merozoites and dispensable or even inhibitory for the naturally flattened sporozoites. Therefore, genetic data indicate a model where AMA1 and the RON proteins have separate roles during apicomplexan invasion. AMA1 acts in a host cell-binding step that impacts the frequency but not quality of RON-dependent TJ formation, and the AMA1–RON2 interaction is not involved in the transduction of the force generated by the zoite motor during invasion. It can be argued that our data are still compatible with an essential function of AMA1 at the TJ, if the residual invasion capacity of AMA1 mutants is ensured by an AMA1-like, functionally redundant protein. This hypothesis is highly unlikely for several reasons. First, the invasive AMA1 mutants displayed a normal entry phenotype including a fully functional TJ. This implies that any compensatory mechanism would need to be of optimal efficiency but expressed in only a subset of mutants (those that invade), a situation different from classical compensation by a suboptimal homolog that affects phenotype quality in all mutant parasites. Second, P. berghei AMA1KD sporozoites generated by Flp/FRT-mediated 3′-untranslated region (UTR) excision and T. gondii AMA1KD tachyzoites generated by Tet-mediated transcriptional repression were silenced immediately before phenotype analysis, thus precluding any selection of compensatory mechanism(s). Interestingly, AMA1KO T. gondii tachyzoites grown in continuous culture, which adapted by overexpressing the AMA1 homologue TgME49_300130 by ~15-fold (Fig. 1d), displayed a significantly milder adhesion phenotype, suggesting that the AMA1 homologue indeed compensated the adhesion defect of AMA1 mutants. The hypothesis of compensation at the TJ is also highly improbable in Plasmodium, which contains a single AMA1 gene. The parasite product most closely related to AMA1 is the transmembrane protein MAEBL40, which in P. berghei is only detected in oocyst sporozoites where it confers binding to the mosquito salivary glands but not invasion of hepatocytes41. Therefore, rather than AMA1-complementing TJ components, AMA1-related proteins in both Toxoplasma and Plasmodium appear to function in zoite adhesion, like AMA1. One question raised by the model in which AMA1 and the RON proteins have dissociated functions is the role of the AMA1–RON2 interaction. The interaction is not essential but is important, being evolutionarily conserved. It might be required for processing/cleavage of surface AMA1 passing the TJ, perhaps allowing the disengagement of interaction of AMA1 with its host cell receptor and facilitating zoite sliding free into the PV. Interestingly, AMA1 undergoes a conformational change upon RON2 binding17, which could lead to loss of adhesive function or exposure of cleavage sites. This would reconcile the genetic data and the fact that antibodies or small molecules that inhibit the interaction can reduce zoite invasion9 10 14 15 16 18 24 42. The increased frequencies of Plasmodium merozoite (relative to adhesive parasites) and sporozoite invasion might also point to a modulatory/inhibitory role, possibly in preventing other interactions important for TJ formation. More work is needed to understand the exact contribution of the AMA1–RON2 interaction, which appears to impact AMA1 but not the TJ per se. The demonstration of the dispensability of AMA1 in any step of host cell invasion by apicomplexan zoites does not question the potential efficacy of AMA1 as target of malaria prevention measures. A large body of work shows the efficacy of antibodies to AMA1 in blocking erythrocyte infection43 44 45, which might also reduce sporozoite invasion of hepatocytes46. Likewise, although the AMA1–RON2 interaction might not have any positive role in invasion, its inhibition by small molecules might still efficiently perturb invasion. Nonetheless, our finding that AMA1-less variants would be only partially impaired in adhesion while retaining normal if not increased invasive capacity, raises the possibility of rapid parasite adaptation to intervention strategies targeting only AMA1 or the AMA1–RON2 interaction. Methods Parasites P. berghei WT ANKA strain GFP fluorescent (GFP@HSP70)36, RFP fluorescent (L733)35, AMA1/Cond27 or AMA1KO were maintained in 3-week-old female Wistar rats or 3-week-old female Swiss mice. Mice or rats were infected with P. berghei parasites by intraperitoneal or intravenous injections. Parasitemia was followed daily by blood smears and FACS analysis. Anopheles stephensi (Sda500 strain) mosquitoes were reared at the Centre for Production and Infection of Anopheles (CEPIA) at the Pasteur Institute as described47. HepG2 cell for sporozoite infection were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or McCoy’s 5A medium supplemented with 10% fetal calf serum and neomycin (50 μg ml−1, Sigma). T. gondii tachyzoites were cultured in human HFF cells maintained in DMEM supplemented with 10% fetal calf serum, 2 mM glutamine and 25 μg ml−1 gentamicin. All experiments using rodents were performed in accordance with the guidelines and regulations of the Pasteur Institute and are approved by the Ethical Committee for Animal Experimentation. Cloning of DNA constructs To generate the plasmid pGFP-hDHFR-PbAMA1KO, the 3′UTR of Pbama1 was amplified from P.berghei genomic DNA (gDNA) with primers 3′UTR PbAMA1 fw/rv and cloned in sites PstI and XhoI in a modified pUC18 plasmid containing a new multiple cloning site and a human dihydrofolate reductase (hDHFR) cassette48 (plasmid BGP-F). The 5′UTR of Pbama1 was amplified with primers 5′UTR PbAMA1 fw/rv and cloned in sites SacI and EcoRI in a pUC18 plasmid containing GFP@HSP70 cassette36 in sites SalI and SacI. Finally, 3′UTR-hDHFR was removed from the previous plasmid and cloned in the latter in sites PstI and SalI. To generate p5RT70-loxPAMA1loxP-YFP-HXGPRT, the TgAMA1 open reading frame (ORF) was amplified from T. gondii gDNA using primers TgAMA1 ORF fw/rv. In addition, 5′UTR and 3′UTR of ama1 was amplified from T. gondii gDNA using 5′UTR TgAMA1 fw/rv or 3′UTR TgAMA1 fw/rv, respectively. First, the 5′UTR of TgAMA1 was inserted upstream of p5RT70 in p5RT70-loxPKillerRedloxP-YFP-HXGPRT plasmid33 in ApaI restriction sites. Then, the killerRed ORF was exchanged by TgAMA1 ORF using EcoRI and PacI. Finally, the 3′UTR of TgAMA1 was cloned in after HXGPRT selection cassette using SacI restriction sites. Transfections and selection P. berghei genetic manipulation was performed as described49. P. berghei AMA1KO were generated by double homologous recombination to replace the endogenous ama1-coding sequence by a hDHFR cassette48 and a GFP fluorescence cassette36. The targeting sequence with the two homologous regions flanking the selection cassettes was PCR amplified from plasmid pGFP-hDHFR-PbAMA1KO using primers 5′UTR PbAMA1 fw and 3′UTR PbAMA1 rv, and gel purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) following kit instructions. After transfection of an enriched preparation of P. berghei ANKA schizonts and re-injection into mice, mutants were selected with constant treatment with pyrimethamine in drinking water until green fluorescent parasitemia was detected. Drugs were used as described49. The presence of AMA1KO was confirmed by PCR analysis with primers Pa/Pb, specific for the WT ama1 locus, and Pb/Pc, specific for integration at the ama1 locus, and by Southern blotting of total gDNA after digestion with the restriction enzymes MfeI or NdeI, with a probe hybridizing at the 5′UTR of ama1, amplified with primers 5′Pbama1-probe fw/rv, to recognize the WT or the mutant loci with different sizes. For T. gondii genetic manipulation, ca 1 × 107 of freshly lysed parasites were transfected with 60 μg linearized DNA by electroporation. Selection was performed with mycophenolic acid (12.5 mg ml−1 in MeOH) and xanthine (20 mg ml−1 in 1 M KOH)50, or phleomycin (50 μg ml−1)51. The TgAMA1IoxP strain was generated by replacement of the endogenous ama1 by floxed ama1 via homologous recombination. The targeting sequence p5RT70-loxPAMA1loxP-YFP-HXGPRT was removed from plasmid by digestion with NsiI and XmaI restriction enzymes and transfected into ku80::diCre recipient strain33. Parasites with stable integration were selected by the treatment with xanthine and mycophenolic acid. Integration by homologous recombination was confirmed by 5′TgAMA1out fw (P1) and p5RT70 rv (P1′) primers. In addition, a PCR with TgAMA1int fw (P2) and TgAMA1 ORF rv (P2′) primers was conducted to discriminate the presence of genomic or coding sequence of ama1 ORF. To generate TgAMA1KO, amal ORF was excised from the genome by activation of diCre with 50 nM rapamycin for 16 h. Subsequent limited dilution of induced TgAMA1loxP pool led to a clonal TgAMA1KO population, which was confirmed by genomic PCR using primers upstream and downstream of the loxP sites, 5′UTR TgAMA1 fw (P3) and YFP rv (P3′). The loss of ama1 ORF was further verified by a PCR in the ORF with primers TgAMA1int fw (P2) and TgAMA1 ORF rv (P2′). For complementation studies, TgAMA1FLAG 52 was used. Southern and western blotting gDNA from P. berghei and T. gondii to use as a PCR template and for Southern blotting was extracted using Qiagen dneasy blood and tissue kit according to manufacturer’s protocol. For Southern blotting of P. berghei gDNA, samples were digested with MfeI or NdeI restriction enzymes overnight, precipitated with ethanol, washed and separated in agarose gel. The gel was transferred to a Hybond-XL membrane (GE-Healthcare) and blotting was performed using the DIG easy Hyb kit and DIG wash and block buffer kit from Roche according to manufacturer’s protocol. The probe was amplified with primers 5′Pbama1-probe fw/rv using the DIG Probe Synthesis kit from Roche. Tachyzoite western blot samples were obtained by spinning down extracellular parasites and incubating with RIPA buffer (50 mM Tris-HCl pH 8; 150 mM NaCl; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) for 20 min on ice. Unless indicated otherwise 106 parasites were loaded onto a SDS acrylamide gel and immunoblot was performed as previously described53. Briefly, proteins were transferred onto a nitrocellulose membrane, after blocking the membranes were incubated with primary antibody for 1 h (mouse anti-AMA1 1:1,000; rabbit anti-aldolase 1:10,000) followed by incubation with horseradish peroxidase-labelled secondary antibodies (1:50,000; Jackson ImmunoResearch) for 2 h. Quantitative PCR RNA from freshly egressed parasites was purified using Trizol followed by chloroform extraction. For cDNA synthesis, 2.5 μg of total RNA were retrotranscribed using the SuperScript VILO (Invitrogen, Life Technology). Quantitative real-time PCR was performed on a LightCycler 480 (Roche) using the LightCycler 480 SYBR Green I Master Mix (Roche). PCR primers were designed to amplify a 100-bp target gene fragment: AMA1 Fw: 5′-TGGAGAGAACCCAGATGCGTTCCT-3′; AMA1 Rv: 5′-CAGTGTAGTCGAGGCAACGGCC-3′; TgME49_300130 Fw: 5′-CCAGGACACGATGCCGCTCG-3′; TgME49_300130 Rv: 5′-AACCCCTCCGCCTCGTCCTT-3′. cDNA levels were normalized to α-tubulin levels measured with primers: Fw: 5′-GCATGATCAGCAACAGCACT-3′; Rv: 5′-ACATACCAGTGGACGAAGGC-3′. Experiments were performed four times with two different RNA preparations. Immunofluorescence For P. berghei merozoites, sporozoites and infected HepG2 cells immunofluorescence, samples were fixed with 4% paraformaldehyde, 0.0075% glutaraldehyde in PBS for 1 h54, permeabilized with 0.1% Triton X-100 in PBS, blocked with BSA 3% in PBS, and stained with primary rabbit polyclonal antibodies to the P. berghei AMA1 peptide CRASHTTPVLMQKPYY (Eurogentec, 1:500 dilution), or primary polyclonal antibodies to the P. berghei RON2 peptide KKLGKLREKIVNGLFKKRGK (Thermo Scientific, 1:500 dilution), followed by secondary Alexa-Fluor-conjugated antibodies (Molecular Probes, 1:500 dilution). Images were acquired using an Axiovert II fluorescence microscope (Zeiss) or the ImageStreamX from AMNIS. For T. gondii immunofluorescence analysis, infected HFF monolayers grown on coverslips were fixed in 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization (0.2% Triton X-100 in PBS) and blocking (2% BSA and 0.2% Triton X-100 in PBS). The staining was performed using primary antibody (mouse anti-AMA1, 1:1,000; mouse anti-SAG1, 1:1,000; rabbit anti-MIC2, 1:500; rabbit anti-IMC1, 1:1,500; rabbit anti-GAP45 1:1,000) followed by secondary Alexa-Fluor-conjugated antibodies (Molecular Probes, 1:3,000). Images were acquired with CCD camera under Deltavision Core or confocal Nikon Ti eclipse microscopes (z–stacks of 0.2–0.3 μm, × 100 immersion objective), deconvolved using SoftWoRx Suite 2.0 (Applied Precision, GE) when needed and further processed using ImageJ 1.34r and Photoshop (Adobe Systems) software. Production of merozoites and ImageStream analysis Erythrocytic merozoites were obtained by culturing infected rat or mouse blood for 16 h, at 37 °C, 5% CO2 and 10% O2, under shaking (90 r.p.m.), in RPMI 1,640 medium (Gibco) supplemented with 20% fetal calf serum and 50 μg ml−1 neomycin. Mature schizonts were separated in a Nycodenz gradient and merozoites were isolated by filtration of schizonts through a 5-μm filter, followed by another filtration through a 1.2-μm filter. The GFP fluorescent AMA1KD strain conditionally knocks down AMA1 expression in mosquito stages, producing sporozoite populations in mosquito salivary glands in which up to 95% of the parasites express undetectable levels of AMA1 (ref. 27). AMA1KD sporozoites were used to infect HepG2 cell in vitro and 62 h post infection the emerging merosomes were collected, and hepatic merozoites were obtained by filtration through a 5-μm filter. The purified merozoites were cultured in vitro with PKH26-stained rat red blood cells for 3 or 10 min under agitation (400 r.p.m.), and cultures were fixed with 4% paraformaldehyde, 0.0075% glutaraldehyde in PBS for further permeabilization and staining with anti-AMA1 (Eurogentec, peptide CRASHTTPVLMQKPYY) and secondary Alexa-Fluor 647. Cells were acquired in an ImageStreamX using a × 60 objective, excitation lasers 488, 561 and 642 nm, and analysed using the software IDEAS, from AMNIS. Merozoites attached to red blood cells were assessed by double fluorescence (PKH26 and GFP), and invaded cells were assessed with a sequence of algorithms that identify PKH26 duplication because of PV formation (R3 Bright Similarity Channels 2 and 4, Intensity Weighted). T. gondii invasion/attachment assay To investigate the attachment and invasion rates of the TgAMA1KO parasites, a red/green invasion assay was performed as described earlier55. HFFs were grown on coverslips of a 24-well plate and infected with 5 × 106 freshly collected parasites. Plates were centrifuged for 2 min at 200 g and incubated at 37 °C, 5% CO2 for 15 min. Subsequently, cells were fixed in 4% paraformaldehyde (PFA) for 15 min followed by immunostaining with α-SAG-1 primary and Alexa-Fluor secondary antibodies before Triton X-100 permeabilization (0.2% in PBS) and immunostaining with α-IMC1 primary and Alexa-Fluor secondary antibodies. Extracellular and intracellular parasites were counted in ten fields of view (× 100 objectives) and calculated as a percentage value of RH Δhxgprt parasites normalized to 100%. To measure T. gondii tachyzoite position relative to the host cell, we adapted the assay previously described27. HeLa cells were plated on poly-lysine-coated glass coverslips in a 6-well plate, transfected with 1 μg of plasmid-encoding mCherry in the pDisplay Vector (Invitrogen) and used 20 h later for a 5-min invasion assay. Cells were fixed in PBS-4% PFA (20 min, room temperature) and stained with anti-SAG-1 antibodies followed by Alexa-Fluor anti-mouse antibodies to label extracellular parasites. Samples were scanned on the confocal Nikon Ti Eclipse microscope and images were captured and analysed with Metamorph software (using the 4D viewer application). For each zoite, an ellipsoid was fit to measure the longitudinal axis, whereas the cell surface contacting the tachyzoite centres of mass was affected to all the isosurfaces. The plane of the cell was reconstructed and angle values between the longer axis of the parasite and the host cell plane were generated by Metamorph. T. gondii replication assay 1 × 105 ku80::diCre or TgAMA1FLAG or 5 × 105 TgAMA1KO were inoculated onto a confluent monolayer of HFFs grown on coverslips (24-well plate) and incubated in normal growth conditions. One hour post inoculation, coverslips were washed in PBS to remove extracellular parasites and thus synchronize the cell cycle. Cells were further grown in normal growth conditions until as indicated, fixed and immunostained. The number of parasites per vacuole was determined for 100 vacuoles. T. gondii plaque assay 200 RH Δhxgprt or TgAMA1FLAG parasites or 1,000 TgAMA1KO parasites were added onto a confluent monolayer of HFF cells of a six-well plate. After incubating for 6 days, the HFF monolayer was washed in PBS and fixed in ice-cold methanol for 20 min. Afterwards, the HFF cells were stained with Giemsa. The area of ten plaques was assessed using Image J 1.34r software. T. gondii egress assay 4 × 105 parasites were grown in HFF monolayers on coverslips for 36 h. Media were exchanged for pre-warmed, serum-free DMEM supplemented with calcium ionophore 2 μM (A23187 in DMSO)56. After incubation for 5 min at normal growth conditions (37 °C; 5% CO2), cells were fixed and stained with anti-GAP45 primary antibody and Alexa-Fluor secondary antibody. Two hundred vacuoles were scored in each experiment. T. gondii motility assay Freshly egressed tachyzoites were allowed to glide for 30 min on glass coverslips coated with 50 μg ml−1 heparin in PBS. Parasites and trails were then stained with anti-P30 antibodies and visualized with an inverted laser scanning microscope (Eclipse Ti, Nikon). Images were analysed using Metamorph and ImageJ software. Numbers of helical and circular trails associated with parasites were scored in 30 fields. T. gondii video microscopy Time-lapse video microscopy was conducted with the DeltaVision® Core microscope using a × 40 immersion lens. Freshly lysed RH Δhxgprt, ku80::diCre or TgAMA1KO were added onto HFF, HeLa or U373 monolayer grown in glass dishes (ibidi; μ-Dish35 mm, high). Forty hours post inoculation, invasion of freshly egressed parasites was observed. Normal growth conditions were maintained throughout the experiment (37 °C; 5% CO2). Images were recorded at one frame per second. Further image processing was performed using ImageJ 1.34r software and with Photoshop (Adobe Systems). Author contributions D.Y.B. and S.T. designed and performed the experiments with Plasmodium, N.A., V.L., J.A.W. and I.T. designed and performed the experiments with Toxoplasma. I.T., M.M. and R.M. conceived this study and designed experiments. D.Y.B., N.A., V.L., I.T., M.M. and R.M. wrote the paper. Additional information How to cite this article: Bargieri, D. Y. et al. Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nat. Commun. 4:2552 doi: 10.1038/ncomms3552 (2013). Supplementary Material Supplementary Movie 1 Time-lapse series showing a RH ku80::diCre tachyzoite invading a HeLa cell. Frames were recorded every 1s and the video is played at 5 frames s-1. The white arrow indicates the TJ. Supplementary Movie 2 Overlay of DIC + GFP time-lapse series showing an AMA1KO tachyzoite invading a HeLa cell. Frames were recorded every 1s and the video is played at 5 frames s-1. The white arrow indicates the TJ. Supplementary Movie 3 Overlay of DIC + GFP time-lapse series showing an AMA1KO tachyzoite invading a U373 cell. Frames were recorded every 1s and the video is played at 5 frames s-1. The white arrow indicates the TJ.

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Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2013

Publication date (Electronic): 13 December 2013

Volume: 8

Issue: 12

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[1 ]National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

[2 ]Department of Parasitology, Faculty of Veterinary Medicine, Sadat City University, Minoufiya, Egypt

[3 ]Department of Animal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Sadat City University, Minoufiya, Egypt

Federal University of São Paulo, Brazil

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* E-mail: igarcpmi@ 123456obihiro.ac.jp

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: MAT JR AU NY II. Performed the experiments: MAT JR AS M. AbouLaila M. Asada HA TM AG. Analyzed the data: MAT JR AS. Contributed reagents/materials/analysis tools: MAT YN NY XX II. Wrote the manuscript: MAT AS NY YN XX II.

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Publisher ID: PONE-D-13-28057

DOI: 10.1371/journal.pone.0083305

PMC ID: 3862764

PubMed ID: 24349483

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Date received : 8 July 2013

Date accepted : 2 November 2013

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This study was supported by a grant from the Global COE Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology and by Grants-in-Aid for Scientific Research from the Japan Society for Promotion of Science (JSPS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa

Intracellular parasite invasion strategies.

Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion

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