Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca
-dependent events. Little is known about TPCs in ...unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca
indicator targeted to the apicoplast, we show that Ca
signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca
uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.
Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide ...NPDP or NVDP minor repeats while cross-reacting with NANP central repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and central repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.
Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (Pi) from the ...host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires Pi from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous Pi by exploiting a gradient of Na+ ions to drive Pi uptake across the plasma membrane. The Na+-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na+-H+-ATPase. Toxoplasma expresses one transmembrane Pi transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon Pi limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing Pi and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for Pi supply for Toxoplasma and also for protection against osmotic stresses.
As an obligate intracellular parasite,
Toxoplasma gondii
must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges ...cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether
Toxoplasma
utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in
Toxoplasma
, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of
Toxoplasma
contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in
in situ
proximity ligation assays. Genetic ablation of
cyp450mt
is not tolerated by
Toxoplasma
; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for
mapr
could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.
Membrane contact sites are regions of close apposition between two organelles, typically less than 30 nanometers apart, that facilitate transfer of biomolecules. The presence of contact sites has ...been demonstrated in yeast, plants, and mammalian cells. Here, we investigated the presence of such contact sites in
. In mammalian cells, endoplasmic reticulum-mitochondria contact sites facilitate mitochondrial uptake of Ca
released by the ER-located inositol 1,4,5-trisphosphate receptor (InsP₃R). However, the InsP₃R in trypanosomes localizes to acidocalcisomes, which serve as major Ca
stores in these parasites. In this work, we have used super-resolution structured illumination microscopy and electron microscopy to identify membrane contact sites that exist between acidocalcisomes and mitochondria. Furthermore, we have confirmed the close association of these organelles using proximity ligation assays. Characterization of these contact sites may be a necessary starting point towards unraveling the role of Ca
in regulating trypanosome bioenergetics.
The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related ...organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the
ndoplasmic
eticulum-resident
alcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of
Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.
The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.
Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19.
We ...obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy.
The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury.
Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2.
This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.
Vacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular ...physiology. We studied the Toxoplasma gondii V-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-ATPase subunits localize to the plasma membrane and to acidic vesicles, and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells, and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a role for V-ATPases in regulating virulence pathways.
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•The V-ATPase localizes to the plasma membrane, PLV, and pro-rhoptries•The V-ATPase supports the maturation of rhoptry and microneme proteins•V-ATPase deficiency leads to accumulation of immature rhoptry and microneme proteins•The function of the V-ATPase impacts every step of the Toxoplasma lytic cycle
Stasic et al. characterize the function of the vacuolar proton ATPase in the life cycle of Toxoplasma gondii, a widespread parasite that infects almost one-third of the world’s population. The work presents molecular evidence of the pump’s role in the synthesis of virulence factors of a highly successful pathogen.
Zinc (Zn
) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory ...proteins. Zn
must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn
are harmful to cells. Zn
transporters (ZnTs) play important functions in cells by reducing intracellular Zn
levels by transporting the ion out of the cytoplasm. We characterized a
gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn
transporter in
TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in
assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn
-sensitive
yeast strain partially restored growth in media with higher Zn
concentrations. These results suggest that TgZnT transports Zn
into the PLV and plays an important role in the Zn
tolerance of
extracellular tachyzoites.
is an intracellular pathogen of human and animals.
pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one.
must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn
transporter of
(TgZnT) that is important for parasite growth. TgZnT restored Zn
tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn
We propose that TgZnT plays a role in Zn
homeostasis during the
lytic cycle.
Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both ...vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism
that is related to the recently described η-class CA found in
. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies.
mutant tachyzoites lacking
display a growth and invasion phenotype
and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries.
is an intracellular pathogen that infects humans and animals. The pathogenesis of
is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new
carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries.
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