ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii.

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen that is highly prevalent in the global population. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we used a biochemical fractionation strategy to predict interactions by correlation profiling. To overcome the deficit of high-quality training data in non-model organisms, we complemented a supervised machine learning strategy, with an unsupervised approach, based on similarity network fusion. The resulting combined high confidence network, ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering identifies 93 protein complexes. We identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community.

Macrophage depletion induces edema through release of matrix-degrading proteases and proteoglycan deposition.

Colony-stimulating factor 1 receptor (CSF1R) blockade abates tumor-associated macrophage (TAM) infiltrates and provides marked clinical benefits in diffuse-type tenosynovial giant cell tumors. However, facial edema is a common adverse event associated with TAM elimination in patients. In this study, we examined molecular and cellular events associated with edema formation in mice and human patients with cancer treated with a CSF1R blocking antibody. Extended antibody treatment of mice caused marked body weight gain, an indicator of enhanced body fluid retention. This was associated with an increase of extracellular matrix-remodeling metalloproteinases (MMPs), namely MMP2 and MMP3, and enhanced deposition of hyaluronan (HA) and proteoglycans, leading to skin thickening. Discontinuation of anti-CSF1R treatment or blockade of MMP activity restored unaltered body weight and normal skin morphology in the mice. In patients, edema developed at doses well below the established optimal biological dose for emactuzumab, a CSF1R dimerization inhibitor. Patients who developed edema in response to emactuzumab had elevated HA in peripheral blood. Our findings indicate that an early increase of peripheral HA can serve as a pharmacodynamic marker for edema development and suggest potential interventions based on MMP inhibition for relieving periorbital edema in patients treated with CSF1R inhibitors.

Vaccine-induced CD8 T cells are redirected with peptide-MHC class I-IgG antibody fusion proteins to eliminate tumor cells in vivo.

Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains.

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Biochemical characterization of FIKK8--A unique protein kinase from the malaria parasite Plasmodium falciparum and other apicomplexans.

FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. Here, we report on the biochemical characterization of Plasmodium falciparum FIKK8 (PfFIKK8) and its Cryptosporidium parvum orthologue (CpFIKK) - the only member of the family predicted to be cytosolic and conserved amongst non-Plasmodium parasites. Recombinant protein samples of both were catalytically active. We characterized their phosphorylation ability using an enzymatic assay and substrate specificities using an arrayed positional scanning peptide library. Our results show that FIKK8 targets serine, preferably with arginine in the +3 and -3 positions. Furthermore, the soluble and active FIKK constructs in our experiments contained an N-terminal extension (NTE) conserved in FIKK8 orthologues from other apicomplexan species. Based on our results, we propose that this NTE is an integral feature of the FIKK subfamily.

Accelerated clearance of Plasmodium-infected erythrocytes in sickle cell trait and annexin-A7 deficiency.

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE(2) formation, Ca(2+) permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7(-/-)) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7(-/-) mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7(-/-) mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE(2)-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7(-/-) erythrocytes and thus confers partial protection against malaria in vivo.

The Plasmodium falciparum-induced anion channel of human erythrocytes is an ATP-release pathway.

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 microM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl(-) or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na-ATP (NMDG-Cl pipette solution) shifted the current reversal potential (V (rev)) from -2 +/- 1 to +51 +/- 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V (rev) to significantly larger (p < or = 0.05, two-tailed t test) values (from -3 +/- 1 to +66 +/- 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 +/- 0.1 nS to 0.2 +/- 0.05 nS and further shifted V (rev) to +87 +/- 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 +/- 4 mV suggesting a relative permeability of P (ATP)/P (Cl) approximately 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway.

A high specificity and affinity interaction with serum albumin stimulates an anion conductance in malaria-infected erythrocytes.

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl(-) and lactate outward currents with an EC(50) of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Influence of amitriptyline on eryptosis, parasitemia and survival of Plasmodium berghei-infected mice.

Plasmodia express a sphingomyelinase, which is apparently required for their development. On the other hand, the sphingomyelinase product ceramide has previously been shown to delay parasite development. Moreover, ceramide triggers suicidal erythrocyte death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Accelerated eryptosis of infected erythrocytes is considered to clear infected erythrocytes from circulating blood and, thus, to favourably influence the clinical course of malaria. The present experiments explored whether the sphingomyelinase inhibitor amitriptyline or genetic knockout of host acid sphingomyelinase influence in vitro parasite growth, eryptosis of Plasmodium falciparum-infected human erythrocytes, in vivo parasitemia and survival of P. berghei-infected mice. Phosphatidylserine exposure was determined by annexin V-binding and cell volume by forward scatter in FACS analysis. In vitro infection of human erythrocytes increased annexin- binding, an effect blunted in the presence of amitriptyline (>or=50 microM). Amitriptyline did not significantly alter intraerythrocytic parasite development but significantly (>or= 1 microM) delayed the increase in parasitemia in vitro. Most importantly, amitriptyline treatment (1 mM in drinking water) resulted in a significant delay of parasitemia and death of infected mice. However, upon infection, ceramide formation was stimulated in both, acid sphingomyelinase knockout mice (Smpd1(-/-)) and their wild type littermates (Smpd1(+/+)). Parasitemia following P. berghei infection was significantly lower in Smpd1(-/-) than in Smpd1(+/+) mice but did not significantly extend the life span of infected animals. In conclusion, mammalian and parasite sphingomyelinase contribute to ceramide formation during malaria, whereby the parasite sphingomyelinase ultimately determines the course of the infection. Amitriptyline presumably blocks both sphingomyelinases and, thus, its use might be a novel strategy to treat malaria.

JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei.

Synthesis of the modified thymine base, beta-d-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de novo site-specific localization of J synthesis within bloodstream form trypanosome DNA. Consistent with this model, we now show that JBP2 (-/-) bloodstream form trypanosomes contain five-fold less base J and are unable to stimulate de novo J synthesis in newly generated telomeric arrays.

Purinoceptors are involved in the induction of an osmolyte permeability in malaria-infected and oxidized human erythrocytes.

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non-infected or non-oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch-clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y1 protein. Moreover, P2Y1-deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection- or oxidation compared with wild-type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y1-deficient mice in vivo compared with wild-type animals. In conclusion, induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves autocrine purinoceptor signaling.

Permselectivity and pH-dependence of Plasmodium falciparum-induced anion currents in human erythrocytes.

Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nutrients and disposal of waste products across the host erythrocyte membrane. Recent patch-clamp experiments have demonstrated inwardly and outwardly rectifying anion conductances in infected but not in control erythrocytes. A ClC-2-generated fraction of the inwardly rectifying current is activated by cell swelling and presumably subserves host cell volume regulation. In contrast, the outwardly rectifying current is insensitive to cell volume but allows the passage of lactate and is involved in the transport of nutrients. The present study was performed to characterize the permselectivity and pH sensitivity of the anion conductances using whole-cell recording. The outwardly rectifying and the inwardly rectifying currents exhibited permselectivities of Cl- > or = Br- approximately I- > SCN- and SCN- > I- > Br- > Cl-, respectively, as evident from the reversal potentials recorded under biionic conditions. While the inwardly rectifying current was not affected significantly by alterations of pH between 6.0 and 8.4, the outward rectifier was inhibited strongly by alkalinization to pH > or = 7.8. Fluxes of 14C-lactate and parasite growth were decreased markedly by the increase of bath pH, an effect that may at least in part be due to inhibition of the outward rectifier and subsequently impaired transport across the erythrocyte membrane.

Artemisinin inhibits cation currents in malaria-infected human erythrocytes.

BACKGROUND: Previous patch-clamp studies have demonstrated inwardly and outwardly rectifying anion currents, ClC-2 Cl- currents, and nonselective Ca(++)-permeable cation currents in Plasmodium falciparum-infected human erythrocytes. METHODS: The current work studied the effect of the potent antimalarial drug artemisinin on the P falciparum infection-induced whole cell currents in human erythrocyte. RESULTS: Artemisinin had no significant effect on the outwardly rectifying anion currents but inhibited the cation-selective currents with an apparent half-maximal inhibitory concentration of < or =10 micromol/L. CONCLUSION: Because artemisinin reportedly inhibits the asexual parasite amplification with much higher potency, the antimalarial action of the drug cannot be attributed to the artemisinin effect on the cation currents. However, artemisinin may be used as a pharmacologic tool to dissect different current fractions in P falciparum-infected erythrocytes.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte.

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Channel-induced apoptosis of infected host cells-the case of malaria.

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca2+-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca2+ and Na+, both of which are required for intracellular growth of the pathogen. The entry of Ca2+ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte "apoptosis" that may represent a host defence mechanism serving to eliminate infected erythrocytes.

Organic osmolyte permeabilities of the malaria-induced anion conductances in human erythrocytes.

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl(-) conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (P(lactate)/P(Cl) = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC(50)s in the range of 0.1 and 1 microM, respectively. In contrast, the IC(50)s of NPPB and furosemide for the inward-rectifying current were >10 microM. Osmotic cell-shrinkage inhibited the inwardly but not the outwardly rectifying conductance. In conclusion, the parasite-induced outwardly-rectifying anion conductance allows permeation of lactate and neutral carbohydrates, whereas the inward rectifier seems largely impermeable to organic solutes. All together, these data should help to resolve ongoing controversy regarding the number of unique channels that exist in P. falciparum-infected erythrocytes.

Dependence of Plasmodium falciparum in vitro growth on the cation permeability of the human host erythrocyte.

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K+ by Na+ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca2+ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na+. The increase of infected cells was further inhibited by EIPA (IC50< 10 microM) and at low extracellular free Ca2+. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM T-butylhydroperoxide for 15 min) or to Ca2+ ionophore ionomycin (1 microM for 60 min). The observations indicate that parasite growth requires the entry of both, Na+ and Ca2+ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca2+ entry further induces break-down of the phospholipid asymmetry in the host membrane.

Electrophysiological properties of the Plasmodium Falciparum-induced cation conductance of human erythrocytes.

Intraerythrocyte survival of the malaria pathogen Plasmodium Falciparum depends on the induction of the new-permeability-pathways (NPPs) in the host cell membrane. NPPs are characterized as anion- and organic osmolyte-permeable channels which also exhibit a low but significant permeability for inorganic cations. To disclose the electrophyiologial properties of this infection-induced cation permeability whole-cell currents were recorded in Plasmodium Falciparum-infected human erythrocytes (pRBC) using bath and pipette solutions with low Cl(-) concentrations. The data disclose a nonselective cation conductance (G(CAT)) which activated upon removal of extracellular Cl(-). Upon activation, G(CAT) was 0.3 +/- 0.05 nS (n=16) in control RBC and 2.0 +/- 0.3 nS (n = 32) in pRBC indicating an induction of G(CAT) during the infection. G(CAT) of pRBC exibited a relative permselectivity for monovalent cations of Cs(+)ñK(+)>Na(+)>Li(+) (P(Na)/P(K) ñ 0.5) with a significant permeability for Ca(2+). G(CAT) of pRBC was inhibited by NPPs blockers (furosemide and NPPB) and cation channel blockers (amiloride, EIPA, GdCl(3)) with the highest sensitivity to EIPA (IC(50)-0.5 microM). Most importantly, the blocker sensitivities differed between the infection-induced anion conductances and G(CAT) suggesting that G(CAT) and the anion conductances represent different channel proteins which in concert build up the NPPs.