The Toxoplasma gondii calcium‐dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival

Apicomplexan parasites express various calcium‐dependent protein kinases (CDPKs), and some of them play essential roles in invasion and egress. Five of the six CDPKs conserved in most Apicomplexa have been studied at the molecular and cellular levels in Plasmodium species and/or in Toxoplasma gondii parasites, but the function of CDPK7 was so far uncharacterized. In T. gondii, during intracellular replication, two parasites are formed within a mother cell through a unique process called endodyogeny. Here we demonstrate that the knock‐down of CDPK7 protein in T. gondii results in pronounced defects in parasite division and a major growth deficiency, while it is dispensable for motility, egress and microneme exocytosis. In cdpk7‐depleted parasites, the overall DNA content was not impaired, but the polarity of daughter cells budding and the fate of several subcellular structures or proteins involved in cell division were affected, such as the centrosomes and the kinetochore. Overall, our data suggest that CDPK7 is crucial for proper maintenance of centrosome integrity required for the initiation of endodyogeny. Our findings provide a first insight into the probable role of calcium‐dependent signalling in parasite multiplication, in addition to its more widely explored role in invasion and egress.     

Arabidopsis dynamin‐related protein 1E in sphingolipid‐enriched plasma membrane domains is associated with the development of freezing tolerance

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin‐related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol‐enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye‐labelled plasma membrane, providing evidence that DRP1E localizes non‐uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol‐enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.     

A novel actin-related protein is associated with daughter cell formation in Toxoplasma gondii

Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.     

Toxoplasma gondii autophagy‐related protein ATG9 is crucial for the survival of parasites in their host

Autophagy is a conserved, life‐promoting, catabolic process involved in the recycling of nonessential cellular components in response to stress. The parasite Toxoplasma gondii is an early‐diverging eukaryote in which part of the autophagy machinery is not exclusively involved in a catabolic process but instead has been repurposed for an original function in organelle inheritance during cell division. This function, depending essentially on protein TgATG8 and its membrane conjugation system, is crucial for parasite survival and prevented an in depth study of autophagy in the mutants generated so far in Toxoplasma. Thus, in order to decipher the primary function of canonical autophagy in the parasites, we generated a cell line deficient for TgATG9, a protein thought to be involved in the early steps of the autophagy process. Although the protein proved to be dispensable for the development of these obligate intracellular parasites in vitro, the absence of TgATG9 led to a reduced ability to sustain prolonged extracellular stress. Importantly, depletion of the protein significantly reduced parasites survival in macrophages and markedly attenuated their virulence in mice. Altogether, this shows TgATG9 is important for the fate of Toxoplasma in immune cells and contributes to the overall virulence of the parasite, possibly through an involvement in a canonical autophagy pathway.     

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.     

An apical protein,Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.     

The cathepsin B of Toxoplasma gondii,toxopain-1, is critical for parasite invasion and rhoptry protein processing

Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative micro1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.     

A coiled‐coil protein is required for coordination of karyokinesis and cytokinesis in Toxoplasma gondii

Toxoplasma gondii is a unicellular eukaryotic pathogen that belongs to the Apicomplexa phylum, which encompasses some of the deadliest pathogens of medical and veterinary importance. The centrosome is key to the organisation and coordination of the cell cycle and division of apicomplexan parasites. The T. gondii centrosome possesses a particular bipartite structure (outer and inner cores). One of the main roles of the centrosome is to ensure proper coordination of karyokinesis. However, how these 2 events are coordinated is still unknown in T. gondii, for which the centrosome components are poorly described. To gain more insights into the biology and the composition of the T. gondii centrosome, we characterised a protein that resides at the interface of the outer and inner core centrosomes. TgCep530 is a large coiled‐coil protein with an essential role in the survival of the parasite. Depletion of this protein leads to the accumulation of parasites lacking nuclei and disruption of the normal cell cycle. Lack of TgCep530 results in a discoordination between the nuclear cycle and the budding cycle that yields fully formed parasites without nuclei. TgCep530 has a crucial role in the coordination of karyokinesis and cytokinesis.     

Seroprevalence and risk factors associated with Toxoplasma gondii in domestic pigs from Spain

Serum samples from 2970 (1400 sows, 1570 fattening) pigs, from 100 farms in the 10 main swine production regions in Spain were tested for antibodies against T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT 1:25 or higher) were detected in 492 pigs (16.6%, 9.7% in fattening pigs and 24.2% in sows). The herd prevalence was 85.0% (95% CI: 78–92) and within-farm prevalence ranged from 2.9% to 92.8% (median = 17.6%). Statistically significant differences were observed among sampling regions with seroprevalence significantly higher in pigs from Valencia Community (27.3%), Extremadura (23.3%) and Catalonia (21.2%). A generalized estimating equations model indicated that the risk factors associated with T. gondii seroprevalence were: age, sows compared to fattening pigs (OR = 2.9; 95% CI = 1.83–4.53), lack of rodent control (OR = 1.9; 95% CI = 1.04–3.60) and presence of cats (OR = 1.6; 95% CI = 1.12–2.34). The seroprevalence observed in the present study indicates a widespread, although variable, exposure to T. gondii among domestic pigs in Spain, which might have important implications for public health. Management measures including control of rodents and cats on the farms could help to reduce the observed prevalence levels in Spain.     

The microneme protein MIC4, or an MIC4-like protein, is expressed within the macrogamete and associated with oocyst wall formation in Toxoplasma gondii

The expression and localisation of MIC4, or an immuno-cross reacting MIC4-like protein, was examined in the enteric forms of Toxoplasma gondii using immunocytochemistry. In addition to being located within the micronemes of the merozoites, MIC4 or the MIC4-like protein was present within the macrogamete and was associated with the developing oocyst wall. The macrogamete is characterised by two types of structurally distinct wall forming bodies (WFB1 and 2). However, by immuno-electron microscopy, it was possible to identify two populations of dense granules (WFB1) which appear to form sequentially during macrogamete development. The first granules to form (WFB1a) stained positively with anti-MIC4 and were followed by MIC4 negative granules (WFB1b). During oocyst wall formation, the WFB1a and b sequentially released their contents onto the surface with WFB1a material forming an anti-MIC4 positive outer veil, while the WFB1b forms the electron dense outer layer of the oocyst wall. The inner layer was formed by WFB2. Thus, for the first time, it was possible to identify two populations of dense granules (WFB1a and b) involved in the formation of different parts of the oocyst wall. It was not possible to analyse the contents of macrogametes by western blot to unequivocally identify the antigen recognised by the polyclonal antisera as MIC4.     

Seropositivity and risk factors associated with Toxoplasma gondii infection in wild birds from Spain

Toxoplasma gondii is a zoonotic intracellular protozoan parasite of worldwide distribution that infects many species of warm-blooded animals, including birds. To date, there is scant information about the seropositivity of T. gondii and the risk factors associated with T. gondii infection in wild bird populations. In the present study, T. gondii infection was evaluated on sera obtained from 1079 wild birds belonging to 56 species (including Falconiformes (n=610), Strigiformes (n=260), Ciconiiformes (n=156), Gruiformes (n=21), and other orders (n=32), from different areas of Spain. Antibodies to T. gondii (modified agglutination test, MAT titer ≥1:25) were found in 282 (26.1%, IC(95%:)23.5-28.7) of the 1079 birds. This study constitute the first extensive survey in wild birds species in Spain and reports for the first time T. gondii antibodies in the griffon vulture (Gyps fulvus), short-toed snake-eagle (Circaetus gallicus), Bonelli's eagle (Aquila fasciata), golden eagle (Aquila chrysaetos), bearded vulture (Gypaetus barbatus), osprey (Pandion haliaetus), Montagu's harrier (Circus pygargus), Western marsh-harrier (Circus aeruginosus), peregrine falcon (Falco peregrinus), long-eared owl (Asio otus), common scops owl (Otus scops), Eurasian spoonbill (Platalea leucorodia), white stork (Ciconia ciconia), grey heron (Ardea cinerea), common moorhen (Gallinula chloropus); in the International Union for Conservation of Nature (IUCN) "vulnerable" Spanish imperial eagle (Aquila adalberti), lesser kestrel (Falco naumanni) and great bustard (Otis tarda); and in the IUCN "near threatened" red kite (Milvus milvus). The highest seropositivity by species was observed in the Eurasian eagle owl (Bubo bubo) (68.1%, 98 of 144). The main risk factors associated with T. gondii seropositivity in wild birds were age and diet, with the highest exposure in older animals and in carnivorous wild birds. The results showed that T. gondii infection is widespread and can be at a high level in many wild birds in Spain, most likely related to their feeding behaviour.     

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.     

Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression

Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signalling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short-term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.     

Seroprevalence of Toxoplasma gondii infection and risk factors associated with seropositivity of pregnant women in Korea

A total of 351 serum samples was examined for anti-T. gondii antibody titers; the overall seroprevalence was 3.7%. The Sabin-Feldman dye test (DT), latex agglutination test (LAT), and IgG enzyme-linked immunosorbent assay (IgG ELISA) indicated seroprevalences of 3.7%, 3.4%, and 4.0%, respectively. Compared with the DT, the sensitivities of the LAT and IgG ELISA were 84.6% and 100.0%, respectively, and the specificities of the LAT and IgG ELISA were both 99.7%. An increase in T. gondii seroprevalence with increasing age was detected, but the difference was not significant. The overall seroprevalence of T. gondii-antibody titers in pregnant Korean women was relatively low compared to those of Europeans and Americans. A multivariate analysis of risk factors showed that T. gondii infection was positively correlated with eating raw meat, but was not associated with the consumption of unwashed vegetables, drinking untreated water, a history of raising a cat, or blood transfusion. The consumption of raw or undercooked meat may, therefore, be the main route of T. gondii infection in Korea.     

A MORN1‐associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding

Apicomplexan parasites replicate by several budding mechanisms with two well‐characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two‐hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two‐hybrid. HAD2a has demonstrated enzyme‐activity in vitro, localizes to the nascent daughter buds, and co‐localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex.     

TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2

Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.