Apicomplexa their genomes mechanism of invasion Plasmodium falciparum MSP1 - The Native Antigen Company Apicomplexa – 3 genomes Membrane compartments of the P. falciparum blood stages. (A) The... | Download Scientific Diagram ›three genomes ›nucleus ›mitochondria ›apicoplast › ›three genomes ›nucleus ›mitochondria ›apicoplast › Apicomplexa – 3 genomes Organism Relevance Genome size Number of genes predicted Babesia bovis Cattle pathogen 8.2 Mb 3,671 Cryptosporidium hominis Human pathogen 10.4 Mb 3,994 Cryptosporidium parvum Human pathogen 16.5 Mb 3,807 Eimeria tenella Intestinal parasite of domestic fowl 55-60 Mb Neospora caninum Pathogen for cattle and dogs 62 Mb Plasmodium berghei Rabbit malaria 18.5 Mb 4,900 Plasmodium chabaudi Rodent malaria 19.8 Mb 5,000 Plasmodium falciparum Human pathogen (malaria) 22.9 Mb 5,268 Plasmodium knowlesi Primate pathogen (malaria) 23.5 Mb 5,188 Plasmodium vivax Human pathogen (malaria) 26.8 Mb 5,433 Plasmodium yoelii yoelii Rodent pathogen (malaria) 23.1 Mb 5,878 Theileria annulata Cattle pathogen 8.3 Mb 3,792 Theileria parva Cattle pathogen (African east coast fever) 8.3 Mb 4,035 Toxoplasma gondii Mammal pathogen 63 Mb 8,100 Tetrahymena thermophila Model organism of ciliates 104 Mb 27,000 Apicomplexa Ciliophora ›three genomes ›nucleus ›mitochondria ›apicoplast › Apicomplexa – 3 genomes ›the smallest mitochondrial genome sequenced ›the 5,967 bp mtDNA ›the form of a circular and/or tandemly repeated linear element ›encodes only three mt protein-coding genes (cox1, cox3 and cob) in addition to the large subunit (LSU) and small subunit (SSU) ribosomal RNA (rRNA) genes › › › › › › › › ›the two rRNA genes are highly fragmented with 20 rRNA pieces having been identified ›the mt-genome organization is perfectly conserved among Plasmodium species ›pairwise sequence similarity of complete mt-genome sequences between these species is very high at 84%–99% https://ars.els-cdn.com/content/image/1-s2.0-S0166685113000315-gr2.jpg https://ars.els-cdn.com/content/image/1-s2.0-S0166685113000315-gr2.jpg Plasmodium spp. – mitochondrial genome In Plasmodium out of a total of 34 identified small RNAs, 20 were initially assigned to both rRNA genes (Feagin et al., 1997); seven more were described later. These fragments range from 23 to 200 nucleotides in size whereby 12 correspond to SSU RNAs (totaling 804 nt) and 15 to LSU RNAs (totaling 1,233 nt) (Feagin et al., 2012). Fragments are dispersed throughout the mitogenome, and are coded for in either strand. It is unclear how these small RNA fragments come together to form a ribosome ›phylogenetic analysis and coalescent-based gene flow modeling to a global collection of Plasmodium falciparum mitogenome to infer the demographic history and geographic origins of malaria parasites https://www.biorxiv.org/content/biorxiv/early/2017/05/29/141853/F4.large.jpg?width=800&height=600&c arousel=1 . Plasmodium spp. – mitochondrial genome enslaved Africans were likely the main carriers of P. falciparum mitochondrial lineages into the Americas after the conquest, additional parasites carried by Australasian peoples in pre-Columbian times may have contributed to the extensive diversity of extant local populations of P. vivax. mitogenomes of a large global collection of human malaria parasites to explore how and when Plasmodium falciparum and P. vivax entered the Americas. We found evidence of a significant contribution of African and South Asian lineages to present-day New World malaria parasites with additional P. vivax lineages appearing to originate from Melanesia that were putatively carried by the Australasian peoples who contributed genes to Native Americans. ›evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) ›totaling 5.9 kb that exist as nonrandom concatemers The 21 minimal T. gondii mtDNA sequence blocks. The DNA sequence is represented by a black line, drawn to scale and named with 21 alphabet characters, A to V (there is no “G”). The coordinates of an SB that encodes a cytochrome or rRNA gene fragment are indicated above the black line and the corresponding coordinates of the assembled gene or rRNA fragment are indicated below the gene fragment; the fragments are colored as defined in the key. Portions of sequence block V contribute to both coxI and coxIII but in different orientations. Toxoplasma – mitochondrial genome Remarkably, none of these SBs encodes for a full‐length protein or rRNA (Figure ​(Figure3).3). Rather, sequencing reads showed that non‐random arrangements of multiple SBs encode full‐length cytochrome and rRNA genes (Namasivayam et al., 2021). For example, blocks E, A and T encode cob, whereas blocks V, L, J, B and M encode coxIII. Homoplasmy of the SBs is maintained by an unknown mechanism, but could be explained by the presence of TgMSH1, a protein previously identified as a yeast MutS homolog that is involved in DNA mismatch repair and in maintaining mtDNA stability, (Garbuz & Arrizabalaga, 2017) or by mtDNA intermolecular recombination. In T. gondii, only a few studies have addressed mitochondrial genome transcription and translation, but we know that both processes are active in this parasite. To translate the three protein‐encoding genes encoded within its mtDNA, the parasite must import a complete set of transfer RNAs (tRNAs) from the cytosol, since the mtDNA of T. gondii does not encode any tRNA genes ›non-photosynthetic plastid found in most Apicomplexa ›originated from an alga through secondary endosymbiosis ›there is debate as to whether this was a green or red alga ›surrounded by four membranes within the outermost part of the endomembrane system ›vital to parasite survival ›hosts important metabolic pathways like fatty acid synthesis, isoprenoid precursor synthesis and parts of the heme biosynthetic pathway ›absent in some of apicomlexans (eg. Cryptosporidium) ›apicoplasts' plant-like properties provide a target for herbicidal drugs ›enticing target for antimalarial drugs ›ancestral genome 35 kb size Výsledek obrázku pro apicoplast Výsledek obrázku pro plasmodium apicoplast APICOMPLEXA: apicoplast Apicoplast as a drug target Delayed Death by Plastid Inhibition in Apicomplexan Parasites - ScienceDirect „delayed death“ Kennedy et al., Trends in Parasitol Inhibitors of replication, transcription, or translation of the apicoplast genome lead to defects in apicoplast biogenesis and segregation. Treated parasites can temporarily continue some metabolic functions (particularly isoprenoid biosynthesis in the case of Plasmodium spp.) but divide to form progeny with an absent or defective apicoplast that is unable to support normal metabolism. By contrast, direct attenuation of Plasmodium isoprenoid biosynthesis (e.g., by fosmidomycin) leads to immediate growth inhibition, indicating that apicoplast-supplied isoprenoids must be necessary elsewhere in the Plasmodium cell. The three plausible isoprenoid endpoints are protein prenylation, dolichol for glycosylphosphatidylinositol (GPI) biosynthesis in the endoplasmic reticulum, and the ubiquinone side-chain in the mitochondrion. Resistance to commonly used malaria drugs is spreading and new drugs are required urgently. The recent identification of a relict chloroplast (apicoplast) in malaria and related parasites offers numerous new targets for drug therapy using well-characterized compounds. The apicoplast contains a range of metabolic pathways and housekeeping processes that differ radically to those of the host thereby presenting ideal strategies for drug therapy. Indeed, many compounds targeting these plastid pathways are antimalarial and have favourable profiles based on extensive knowledge from their use as antibacterials. ›Apicoplast genomes are quite similar, suggesting that much of the reduction in coding capacity happened prior to splitting the apicomplexan lineages › ›the apicoplast genome has been under high selective pressure - reducing the genome size ›(chloroplast genomes average 150–200 kb in size, those of non-photosynthetic plants are ~70 kb) ›P. falciparum (Wilson et al., 1996) ›T. gondii (ToxoDB) ›E. tenella (Cai et al., 2003) ›all ~35 kb in size › ›small subunit (SSU) and large subunit (LSU) rRNAs encoded head to head ›separated by seven tRNA genes ›single tRNA gene is found at the 3′ ends of both rRNAs › ›this organization is highly reminiscent of chloroplast genomes › Apicoplast genome 25 copies of the apicoplast genome in T. gondii and 15 in P. falciparum multiple copies of a genome would facilitate repair of mutations by gene conversion Apicoplast genome of Plasmodium ›low complexity and primarily encodes genes involved in its own expression ›one of the most A/T-rich genomes known to date with 86.9% A/T ›contains 68 genes coding for the large and small subunit rRNAs, a minimal but complete set of tRNAs, ribosomal proteins, three subunits of a bacterial-like RNA polymerase, and several protein chaperones https://ars.els-cdn.com/content/image/1-s2.0-S0166685116300792-fx1_lrg.jpg › ›the gene content of the apicoplast genomes is highly conserved apart from a few lineage specific genes › ›SSU and LSU rRNAs (rrs and rrl) › ›three subunits of the bacteria-type RNA polymerase (rpoB, rpoC1, rpoC2) › ›16 ribosomal proteins, an EF-Tu, a ClpC-like protein › ›24 tRNA species, the minimum sufficient for translation without importing a tRNA from the cytosol › › Apicoplast function apicoplast proteins encoded by the nuclear genome ›a bipartite organellar targeting sequence at the N terminus ›more than 500 proteins encoded by the nuclear genome of P. falciparum have an apicoplast targeting sequence › ›enzymes involved in: › de novo biosynthesis of isoprenoid, fatty acid and heme ›housekeeping proteins such as DNA polymerase, DNA gyrase subunits, ribosomal proteins, molecular chaperones ›components of a Suf type Fe–S cluster assembly system Isoprenoid precursor biosynthesis and CoA biosynthesis pathways of Plasmodium falciparum and the utilization of their end products. Isoprenoid precursors IPP and its regioisomer DMAPP generated by apicoplast MEP pathway are utilized both within and outside the apicoplast. Within the organelle, isoprenoid precursors are used for tRNA modification and the synthesis of long-chain polyprenyls, whereas outside the organelle, they are used for protein prenylation in cytosol (black-outlined box), ubiquinone synthesis in the mitochondrion (orange-outlined box), and dolichol synthesis in the endoplasmic reticulum (ER) (blue-outlined box). FeS generated by the FeS synthesis pathway are essential for the activity of two terminal enzymes of MEP pathway. DXPR is the target of Fos. The CoA synthesis pathway begins in the cytosol and ends in the apicoplast. Within the organelle, CoA is used for FASII, while outside the organelle, it is used in the tricarboxylic acid (TCA) cycle and acetyl-CoA synthesis in the mitochondrion (orange-outlined box). Boxes filled in gray are nonessential pathways/processes in the blood-stage parasites. GA3P, glyceraldehyde-3-phosphate; IspD, 2C-methyl-D-erythritol 4-phosphate cytidyltransferase; IspE, 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) kinase; CDP-MEP, CDP-ME phosphate; IspF, 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) synthase; IspG, hydroxylmethylbutenyl diphosphate (HMB-PP) synthase; IspH, HMB-PP reductase; MiaA, tRNA isopentenyltransferase; MiaB, tRNA-i6A37 methylthiotransferase; Pan, pantothenate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; FPP/GGPPS, farnesyl/geranylgeranyl pyrophosphate synthase; PPRD, polyprenol reductase. APICAL COMPLEX only in invading stages „zoit“ repeated dedifferentation and differentation in Apicomplexa life cycle 2 components skeletal and secretory Výsledek obrázku pro apicomplexa ZOIT = infecting stage Výsledek obrázku pro apicoplast toxoplasma INVASION into host cells proteins secreted from apical complex required apical-end zoite orientation Výsledek obrázku pro Alan F. Cowman plasmodium invasion Výsledek obrázku pro apical complex skeletal part Výsledek obrázku pro apical complex conoid conoid is absent in Plasmodium, Babesia and Theileria The alveoli and proteinaceous skeleton form a structure called the inner membrane complex (IMC), which, together with the subpellicular microtubules, provides the shape and stability of the cell. The most apical portion of IMC is APICAL COMPLEX SKELETAL PART OF APICAL COMPLEX Výsledek obrázku pro apical complex conoid SKELETAL PART OF APICAL COMPLEX •preconoidal rings (= apical rings) •conoid = spirally arranged microtubules •one or more polar rings • •MTOC – microtubule organizing center Animation of kinesin "walking" on a microtubule SECRETORY PART OF APICAL COMPLEX Rhoptries ›club-shaped organelles connected by thin necks to the extreme apical pole of the parasite ›enzymes that are released during the penetration process → internalization into the host cell ›egress from the host cell ›proteins to create parasitopholous vacuole and establishment the parasite inside ›modification of the surface of the host cell figure1 Fig.1 Rhoptries + Micronemes + Dense Granules Výsledek obrázku pro apical complex electron microscopy SECRETORY PART OF APICAL COMPLEX Micronemes ›proteins specialized in attachement onto host cell surface receptors and facilitating erythrocyte entry ›only by this initial chemical exchange can the parasite enter into the erythrocyte via actin-myosin motor complex ›motility → TRAP protein Dense granules ›secretion takes place after parasite invasion and localization within parasitophorous vacuole ›persists for several minutes figure1 Výsledek obrázku pro apical complex electron microscopy Výsledek obrázku pro apical complex electron microscopy Cooperative role of microneme and rhoptry proteins for invasion Cooperative roles of microneme and rhoptry proteins for invasion. (a) Rhoptry secretion relies on host cell attachment mediated by recognition of host cell receptors by MICs (left). The two rhoptry necks fuse with the AV and with the PPM. Rhoptry neck proteins are then released and establish complexes with MICs to initiate the formation of a fusion pore in the HPM (middle) and then to build the MJ (right). In Plasmodium falciparum, pore formation may be dependent on formation of a stable complex between two microneme proteins, Ripr and CyRPA, and the rhoptry neck protein Rh5, which assemble at the host-parasite interface to interact with the host receptor basigin. This in turn allows delivery of the rhoptry neck proteins RON2, RON4, and RON5 into the host cell and intimate interaction of the extracellular part of the transmembrane protein RON2 with the micronemal protein AMA1. In Toxoplasma, the RON proteins recruit cytosolic host proteins such as ALIX, TSG101, CD2AP, and CIN85. (b) Close-up of the MJ complex during invasion. The MJ connects the PPM and HPM via the interaction between the ectodomain of AMA1 and the C-terminal region of RON2. On the parasite side, the cytoplasmic tail of AMA1 might be connected to the glideosome machinery. On the host side, actin and tubulin accumulate at the MJ and RONs subvert host protein networks. RON4 possesses two YP(x)nL motifs that specifically recruit two ALIX proteins. RON5 has one PPPY and two P(T/S)AP motifs that account for accumulation of TSG101 at the MJ. Moreover, RON2, RON4, and RON5 contain multiple Px(P/A)xPR motifs that bind to CIN85 and CD2AP and massively recruit CIN85/CD2AP to the MJ. (c) Top view of the cocrystal structures of Toxoplasma gondii AMA1 (purple) with its RON2-interacting peptide (RON2sp) (green). TgRON2sp is anchored into the apical groove of TgAMA1 in a disulfide-anchored U-shaped conformation. Abbreviations: ALIX, apoptosis-linked gene 2–interacting protein X; AMA1, apical membrane antigen 1; AV, apical vesicle; BSG, basigin; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; CyRPA, cysteine-rich protective antigen; HPM, host plasma membrane; MIC, micronemal; MJ, moving junction; PPM, parasite plasma membrane; PV, parasitophorous vacuole; Rh5, reticulocyte-binding protein homolog 5; Ripr, Rh5-interacting protein; RON2, rhoptry neck protein 2; TSG101, tumor susceptibility gene 101. Figure adapted with permission from Reference 147. Cooperative role of microneme and rhoptry proteins for invasion Moving junction (MJ) ›a tight connection between invading parasite and host cell membranes through which the parasite passes to enter into the host › ›AMA1 = apical membrane antigen ›RON2= rhoptry neck protein › ›AMA1 binds to RON2 that is inserted into the host cell membrane at the site of invasion ›the AMA1-RON2 complex contribute to the formation of moving junction (target for vaccines and drugs) ›MJ assembles at the site of parasite invasion and provides a site of traction for active penetration of the host cell and coincident formation of the parasitophorous vacuole Cooperative roles of microneme and rhoptry proteins for invasion. (a) Rhoptry secretion relies on host cell attachment mediated by recognition of host cell receptors by MICs (left). The two rhoptry necks fuse with the AV and with the PPM. Rhoptry neck proteins are then released and establish complexes with MICs to initiate the formation of a fusion pore in the HPM (middle) and then to build the MJ (right). In Plasmodium falciparum, pore formation may be dependent on formation of a stable complex between two microneme proteins, Ripr and CyRPA, and the rhoptry neck protein Rh5, which assemble at the host-parasite interface to interact with the host receptor basigin. This in turn allows delivery of the rhoptry neck proteins RON2, RON4, and RON5 into the host cell and intimate interaction of the extracellular part of the transmembrane protein RON2 with the micronemal protein AMA1. In Toxoplasma, the RON proteins recruit cytosolic host proteins such as ALIX, TSG101, CD2AP, and CIN85. (b) Close-up of the MJ complex during invasion. The MJ connects the PPM and HPM via the interaction between the ectodomain of AMA1 and the C-terminal region of RON2. On the parasite side, the cytoplasmic tail of AMA1 might be connected to the glideosome machinery. On the host side, actin and tubulin accumulate at the MJ and RONs subvert host protein networks. RON4 possesses two YP(x)nL motifs that specifically recruit two ALIX proteins. RON5 has one PPPY and two P(T/S)AP motifs that account for accumulation of TSG101 at the MJ. Moreover, RON2, RON4, and RON5 contain multiple Px(P/A)xPR motifs that bind to CIN85 and CD2AP and massively recruit CIN85/CD2AP to the MJ. (c) Top view of the cocrystal structures of Toxoplasma gondii AMA1 (purple) with its RON2-interacting peptide (RON2sp) (green). TgRON2sp is anchored into the apical groove of TgAMA1 in a disulfide-anchored U-shaped conformation. Abbreviations: ALIX, apoptosis-linked gene 2–interacting protein X; AMA1, apical membrane antigen 1; AV, apical vesicle; BSG, basigin; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; CyRPA, cysteine-rich protective antigen; HPM, host plasma membrane; MIC, micronemal; MJ, moving junction; PPM, parasite plasma membrane; PV, parasitophorous vacuole; Rh5, reticulocyte-binding protein homolog 5; Ripr, Rh5-interacting protein; RON2, rhoptry neck protein 2; TSG101, tumor susceptibility gene 101. Figure adapted with permission from Reference 147. Cooperative role of microneme and rhoptry proteins for invasion Moving junction (MJ) The moving junction, a key portal to host cell invasion by apicomplexan parasites - ScienceDirect ZOIT MOTILITY → „gliding“ locomotion ›a unique machinery called the glideosome ›composed of an actomyosin system that underlies the plasma membrane ›glideosome promotes substrate-dependent gliding motility ›active host cell entry and egress from infected cells ›carefully choreographed and regulated by both internal and external factors ›calcium signaling pathways playing an integral role › › ›anchoring of the motor complex internally so that when the motor is engaged, a locomotory force can be generated that propels the parasite over the substrate › ›the proteins first implicated in directly anchoring the motor were termed gliding associated proteins or GAPs Výsledek obrázku pro apicomplexa gliding ZOIT MOTILITY → „gliding“ locomotion ›establishment of transient contacts with the substrate via molecules of an adhesion complex ›released from the apically positioned microneme organelles into the plasma membrane of the parasite ›most well-characterized of adhesins include the apical membrane antigen-1 (AMA1) protein, and members of the thrombospondin-related anonymous protein (TRAP) family - indirectly link the motor complex to the adhesion site › ›connection of the adhesins to the molecular motor apparatus Scheme of a Toxoplasma gondii tachyzoite with a detailed model of the "glideosome", the molecular machinery promoting gliding motility. Abbreviations: GAP45 and GAP50, gliding associated protein 45 kDa and 50 kDa, respectively; IMC, Inner Membrane Complex; IMP, Inner Membrane Particle, MyoA, myosin A; MLC or MTIP, Myosin light chain or Myosin tail-interacting protein. Working model of the glideosome in Toxoplasma gondii. A myosin light chain (MLC1) facilitates the interaction of Myosin A with GAP45 that spans the space between the inner membrane complex (IMC) and the parasite plasma membrane (PM). This motor complex is anchored to the IMC via GAP50 and GAP 40, with additional stabilization by GAPMs. The IMC is, itself, structurally supported by a subpellicular network consisting of alveolins. Myosin A interacts with short actin filaments that, in turn, are linked to cell surface adhesins (MIC2) via aldolase. The role of aldolase as an intermediary linker is currently under renewed scrutiny. ›TRAP is secreted from micronemes ›capping involves the directional movement of protein complexes from the anterior end of the parasite to the posterior end, where they are shed ›gliding motility is driven by the capping of transmembrane proteins linked to a fixed point on the substrate along a subcortical actinomyosin complex Výsledek obrázku pro parasitophorous vacuole HOST CELL INFECTION: invasion → replication → egress https://www.youtube.com/watch?v=TIc6exbsH90 https://www.youtube.com/watch?v=JSuSsn4HwHI