Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

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.

The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo.

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.

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation.

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.

TbVps41 regulates trafficking of endocytic but not biosynthetic cargo to lysosomes of bloodstream forms of Trypanosoma brucei.

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket ("big eye" phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.

The Rab11-family interacting proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner.

After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma, suggesting that specific parasite PV proteins may be involved in these processes.

Endothelial thrombomodulin downregulation caused by hypoxia contributes to severe infiltration and coagulopathy in COVID-19 patient lungs.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.

A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii.

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-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 Ca2+ indicator targeted to the apicoplast, we show that Ca2+ 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 Ca2+ 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.

Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii.

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing "tried and true" methods of our lab who pioneered the first use of GECI's in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites.

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 Plasmodium falciparumendoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum 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.IMPORTANCE 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.

The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity.

Zinc (Zn2+) 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. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii 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 in vitro 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 Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites.IMPORTANCEToxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii 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 Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+ We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

The Toxoplasma Vacuolar H+-ATPase Regulates Intracellular pH and Impacts the Maturation of Essential Secretory Proteins.

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.

Acidocalcisome-Mitochondrion Membrane Contact Sites in Trypanosoma brucei.

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 Trypanosoma brucei. In mammalian cells, endoplasmic reticulum-mitochondria contact sites facilitate mitochondrial uptake of Ca2+ released by the ER-located inositol 1,4,5-trisphosphate receptor (InsP3R). However, the InsP3R in trypanosomes localizes to acidocalcisomes, which serve as major Ca2+ 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 Ca2+ in regulating trypanosome bioenergetics.

A Glycosylphosphatidylinositol-Anchored Carbonic Anhydrase-Related Protein of Toxoplasma gondii Is Important for Rhoptry Biogenesis and Virulence.

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 Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. 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. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro 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. IMPORTANCEToxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii 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 T. gondii 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.