Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.     

Red cell deformability and invasion by malaria parasites

Malaria parasites enter red cells in a multi-step process involving attachment, membrane deformation, invagination and encapsulation. The molecular basis of red cell rigidity is examined by Geoff Pasvol and lain Wilson, and they discuss its effect on the efficiency of invasion by various Plasmodium spp.     

Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.     

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii .     

Schistosoma mansoni: molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.     

Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.     

The role of natural killer T cells and other T cell subsets against infection by the pre-erythrocytic stages of malaria parasites

T cells are critical mediators of immunity to the pre-erythrocytic stages of malaria parasites. In this review, we survey the role of the various T cell subsets in combating the pre-erythrocytic stages; in particular, the role of NK T cells. Moreover, we show how studies using malaria models have revealed a unique behavior of NK T cells, namely the bridging of innate and adaptive immunity.     

Cyclin A2:At the crossroads of cell cycle and cell invasion

Cyclin A2 is an essential regulator of the cell division cycle through the activation of kinases that participate to the regulation of S phase as well as the mitotic entry. However,whereas its degradation by the proteasome in mid mitosis was thought to be essential for mitosis to proceed,recent observations show that a small fraction of cyclin A2 persists beyond metaphase and is degraded by autophagy. Its implication in the control of cytoskeletal dynamics and cell movement has unveiled its role in the modulation of Rho A activity. Since this GTPase is involved in both cell rounding early in mitosis and later,in the formation of the cleavage furrow,this suggests that cyclin A2 is a novel actor in cytokinesis. Taken together,these data point to this cyclin as a potential mediator of cell-niche interactions whose dysregulation could be taken as a hallmark of metastasis.     

Extracellular Toxoplasma gondii tachyzoites do not require carbon source uptake for ATP maintenance, gliding motility and invasion in the first hour of their extracellular life

Apicomplexan parasites undergo metabolic shifts in adaptation to environmental changes. Here, we investigate the metabolic requirements which are responsible for ATP homeostasis in the extracellular stage of Toxoplasma gondii. Surprisingly, we found that freshly released tachyzoites are able to maintain a constant ATP level during the first hour of extracellular incubation without the acquisition of external carbon sources. We further demonstrated that the extent of gliding motility and that of host cell invasion is independent from the availability of external carbon sources during this one hour extracellular period. The ATP level and the invasion efficiency of extracellular parasites were severely decreased by treatment with the glycolysis inhibitor, 2-deoxy-d-glucose, but not by the F(0)F(1)-ATPase inhibitor, oligomycin. This suggests that although the uptake of glucose itself is not required during the 1h incubation period, extracellular parasites depend on the activity of the glycolytic pathway for ATP homeostasis. Furthermore, active glycolysis was evident by the secretion of lactate into the culture medium, even in the absence of external carbon sources. Together, our studies suggest that tachyzoites are independent from external carbon sources within the first hour of their extracellular life, which is the most relevant time span for finding a new host cell, but rely on the glycolytic metabolisation of internal carbon sources for ATP maintenance, gliding motility and host cell invasion.     

L1CAM: A major driver for tumor cell invasion and motility

The L1 cell adhesion molecule (L1CAM) plays a major role in the development of the nervous system and in the malignancy of human tumors. In terms of biological function, L1CAM comes along in two different flavors: (1) a static function as a cell adhesion molecule that acts as a glue between cells; (2) a motility promoting function that drives cell migration during neural development and supports metastasis of human cancers. Important factors that contribute to the switch in the functional mode of L1CAM are: (1) the cleavage from the cell surface by membrane proximal proteolysis and (2) the ability to change binding partners and engage in L1CAM-integrin binding. Recent studies have shown that the cleavage of L1CAM by metalloproteinases and the binding of L1CAM to integrins via its RGD-motif in the sixth Ig-domain activate signaling pathways distinct from the ones elicited by homophilic binding. Here we highlight important features of L1CAM proteolysis and the signaling of L1CAM via integrin engagement. The novel insights into L1CAM downstream signaling and its regulation during tumor progression and epithelial-mesenchymal transition (EMT) will lead to a better understanding of the dualistic role of L1CAM as a cell adhesion and/or motility promoting cell surface molecule.     

Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

A comprehensive study reveals that kinesins in the malaria parasite Plasmodium have diverse cellular roles and locations, including functions in spindle assembly during proliferation, axoneme formation in flagellum biogenesis, and determining the apical morphology of the cell.     

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.     

Cytochemical study of the different stages in the life cycle of Toxoplasma gondii. IX. The polysaccharides and lipids in the parasites at developmental stages from the cat intestine]

Amylopectin was detected in all the stages examined. In the oval stages the minute granules of PAS-positive material were seen in the cytoplasm when examined on fresh-frozen sections. In merozoites, amylopectin was more conspicuous with maturation. The residual body of microgametocytes contain large amounts of amylopectin; no polysaccharide was visualized in microgamete bodies. Amylopectin was most abundant in macrogametocytes and zygotes. However, no peripheral position of PAS-positive "plastic granules" (wall-forming bodies), so characteristic of other coccidia and revealed by the electron microscopy for T. gondii macrogametocytes, was seen. Acid mucopolysaccharides in the macrogametocyte were detected in the central zone, leaving the periphery of the cell unstained. Very small, if any, amounts of lipids were detected in asexual stages of T. gondii. Unlike, large accumulation of lipid droplets were seen in growing macrogametocytes suggesting the involvement of lipids along with amylopectin in the metabolism of oocysts later discharged from the host body.     

A conserved checkpoint pathway mediates DNA damage--induced apoptosis and cell cycle arrest in C. elegans

To maintain genomic stability following DNA damage, multicellular organisms activate checkpoints that induce cell cycle arrest or apoptosis. Here we show that genotoxic stress blocks cell proliferation and induces apoptosis of germ cells in the nematode C. elegans. Accumulation of recombination intermediates similarly leads to the demise of affected cells. Checkpoint-induced apoptosis is mediated by the core apoptotic machinery (CED-9/CED-4/CED-3) but is genetically distinct from somatic cell death and physiological germ cell death. Mutations in three genes--mrt-2, which encodes the C. elegans homolog of the S. pombe rad1 checkpoint gene, rad-5, and him-7-block both DNA damage-induced apoptosis and cell proliferation arrest. Our results implicate rad1 homologs in DNA damage-induced apoptosis in animals.