Plasmodium berghei histamine‐releasing factor favours liver‐stage development via inhibition of IL‐6 production and associates with a severe outcome of disease

Plasmodium spp., which causes malaria, produces a histamine‐releasing factor (HRF), an orthologue of mammalian HRF. Histamine‐releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre‐erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito‐injected parasite form into the erythrocyte‐infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL‐6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti‐IL‐6 antibodies or infection in IL‐6‐deficient mice and parasite HRF is sufficient to decrease IL‐6 synthesis, indicating a direct role of parasite HRF in reducing host IL‐6. The target cells modulated by HRF for IL‐6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down‐regulate a cytokine with anti‐parasite activity. Our data also highlight the link between a prolonged transition from liver to blood‐stage infection and reduced incidence of experimental cerebral malaria.     

Feeling at home from arrival to departure: protein export and host cell remodelling during Plasmodium liver stage and gametocyte maturation

Obligate intracellular pathogens actively remodel their host cells to boost propagation, survival, and persistence. Plasmodium falciparum, the causative agent of the most severe form of malaria, assembles a complex secretory system in erythrocytes. Export of parasite factors to the erythrocyte membrane is essential for parasite sequestration from the blood circulation and a major factor for clinical complications in falciparum malaria. Historic and recent molecular reports show that host cell remodelling is not exclusive to P. falciparum and that parasite‐induced intra‐erythrocytic membrane structures and protein export occur in several Plasmodia. Comparative analyses of P. falciparum asexual and sexual blood stages and imaging of liver stages from transgenic murine Plasmodium species show that protein export occurs in all intracellular phases from liver infection to sexual differentiation, indicating that mammalian Plasmodium species evolved efficient strategies to renovate erythrocytes and hepatocytes according to the specific needs of each life cycle phase. While the repertoireof identified exported proteins is remarkably expanded in asexual P. falciparum blood stages, the putative export machinery and known targeting signatures are shared across life cycle stages. A better understanding of the molecular mechanisms underlying Plasmodium protein export could assist in designing novel strategies to interrupt transmission between Anopheles mosquitoes and humans.     

THE BIOLOGY OF HARVEYELLA MIRABILIS (CRYPTONEMIALES,RHODOPHYCEAE). VI. TRANSLOCATION OF PHOTOASSIMILATED 14C122

Pulse-chase labelling experiments demonstrate that photoassimilated ¹⁴C-bicarbonate is translocated from the host red alga Odonthalia floccosa (Esper) Falkenberg to the parasite Harveyella mirabilis (Reinsch) Schmitz & Reinke. The primary path of translocation is from host cortical cells (the site of photoassimilation) to the erumpent parasite pustule via the zone of interdigitation. The latter is a tissue region in which rhizoidal cells of Harveyella grow between, and establish secondary pit plugs with medullary cells of Odonthalia. A secondary translocation pathway occurs from isolated host cells dispersed in the pustule of Harveyella to adjacent parasite cells.     

Quantitative Proteomic Analysis of Simian Primary Hepatocytes Reveals Candidate Molecular Markers for Permissiveness to Relapsing Malaria Plasmodium cynomolgi

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long‐term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host–parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR‐BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.     

Calyptospora tucunarensis n. sp. (Apicomplexa: Sporozoea) from the liver of tucunare Cichla ocellaris in Brazil

A coccidian infection of high prevalence and intensity was demonstrated in the liver of tucunare Cichla ocellaris from both natural waters of northeast Brazil and those cultured in fish farms. The immature oöcysts of this parasite were seen uniformly dispersed in the liver,and its mature oöcysts formed large aggregations. The sporocysts were characterised by a single sporopodium and a membranous veil surrounding them. The parasite is described as a new species, Calyptospora tucunarensis. This is the second report on a calyptosporid in a South American fish.     

Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5‐kinase (PIKfyve) that converts phosphatidylinositol 3‐phosphate [PI(3)P] into phosphatidylinositol 3,5‐bisphosphate [PI(3,5)P₂] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non‐pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P₂ effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P₂‐dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.      

Imaging liver‐stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High‐speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver‐stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.     

Plasmodium falciparum polypeptides interacting with human red cell membranes show high affinity binding to Band-3

P. falciparum proteins were labelled with [³⁵S]methionine and harvested at various asexual stages. A number of parasite proteins bound to uninfected red cell membranes (ghosts). Some of these proteins differentially partitioned when ghosts were extracted with detergent. Several of these proteins bound very strongly to immobilised whole ghost proteins or immobilised purified Band-3 in a stage-specific manner, but not to a sham-coupled matrix or to immobilised Band-3 extract from cells rendered refractory to invasion. Such specific binding of parasite proteins to immobilised Band-3 supports recent conjecture as to its role as a host receptor during parasite invasion. However, our results demonstrate the complex and multifactorial nature of the interaction between parasite and host proteins during invasion and development.     

Characterization of Host Cell Mutants Significantly Resistant to Cryptosporidium parvum Infection

Cryptosporidium parvum is a parasitic protist and a causative agent of mild‐to‐severe diarrheal diseases in humans and animals. Despite its globally recognized importance, knowledge on the mechanism of parasite invasion and molecular interactions between host cells and the parasite is limited. Here, we report the establishment of 43 mutant cell lines derived from HCT‐8 cells by UV‐induced mutagenesis and the characterization of three mutants with significantly reduced susceptibility to cryptosporidial infection. Based on qRT‐PCR assay performed at 18 h postinfection time, the parasite loads could be reduced by ~45%, ~35%, and ~20% in mutants A05, B08, and B12, respectively (p < 0.001 in all three mutants vs. HCT‐8 cells). The mutagenesis mainly affected the attachment of parasite in A05 (i.e. ~30% reduction, p < 0.001 vs. HCT‐8), and intracellular development in B08 and B12. The three cell mutants may serve as valuable reagents to further investigate the mechanism of parasite invasion and intracellular development by identifying the gene mutations associated with the parasite attachment (A05) and intracellular development (B08 and B12).     

Observations on Haemogregarina balli sp. n. from the Common Snapping Turtle,Chelydra serpentina*

SYNOPSIS. Haemogregarina balli sp. n. is described from the blood and organs of the common snapping turtle Chelydra serpentina serpentina and from the gastric and intestinal ceca of the presumed invertebrate hosts, the leeches Placobdella parasitica and Placobdella ornata. In the peripheral blood of the turtle, male and female gametocytes and immature erythrocytic schizonts are found within erythrocytes. The maturation of erythrocytic schizonts containing 6–8 merozoites is recorded from liver imprints. Schizonts with 13–25 merozoites are found in various cells of the liver, lung and spleen. In the gastric ceca of the leeches the host erythrocytes are digested, releasing the gametocytes and immature erythrocytic schizonts. Immature erythrocytic schizonts degenerate. Association of the gametocytes occurs in the intestinal ceca. The microgametocyte apparently gives rise to 4 nonmotile microgametes, one of which fertilizes the macrogamete while the other remain as condensed, residual nuclei on the periphery of the developing oocyst. The oocyst increases in size with maturity. A mature oocyst produces 8 sporozoites from a single germinal center. Sporozoites liberated from the oocyst are found in the tissues of the leech. Transovarial transmission of the parasite does not occur in the turtle. Attempts at experimental transmission failed. Previously unfed (control) leeches were negative for the parasite. Haemogregarina balli is compared with other haemogregarines described from C. serpentina. Features of species of Haemogregarina and Hepatozoon as well as the taxonomy of these genera are discussed.     

High resolution microscopy reveals an unusual architecture of the Plasmodium berghei endoplasmic reticulum

To fuel the tremendously fast replication of Plasmodium liver stage parasites, the endoplasmic reticulum (ER) must play a critical role as a major site of protein and lipid biosynthesis. In this study, we analysed the parasite's ER morphology and function. Previous studies exploring the parasite ER have mainly focused on the blood stage. Visualizing the Plasmodium berghei ER during liver stage development, we found that the ER forms an interconnected network throughout the parasite with perinuclear and peripheral localizations. Surprisingly, we observed that the ER additionally generates huge accumulations. Using stimulated emission depletion microscopy and serial block‐face scanning electron microscopy, we defined ER accumulations as intricate dense networks of ER tubules. We provide evidence that these accumulations are functional subdivisions of the parasite ER, presumably generated in response to elevated demands of the parasite, potentially consistent with ER stress. Compared to higher eukaryotes, Plasmodium parasites have a fundamentally reduced unfolded protein response machinery for reacting to ER stress. Accordingly, parasite development is greatly impaired when ER stress is applied. As parasites appear to be more sensitive to ER stress than are host cells, induction of ER stress could potentially be used for interference with parasite development.     

Composite bundles,the host/parasite interface in the holoparasitic angiosperms Langsdorffia and Balanophora (Balanophoraceae)

Composite bundles are not simply a type of vascular bundles, but an integrated host/parasite interface. We investigated their structure in tubers of Langsdorffia and Balanophora. Composite bundles in both genera have similar components: 1) a central mass of host vascular tissues among which are located large parasite transfer cells; 2) a sheath of parasite parenchyma surrounding the central host vascular tissues; 3) specialized conducting tissues in the sheath; and 4) apical meristems composed of both host and parasite meristematic cells. Sheath parenchyma is recognizable from parasite tuber matrix by having thinner cell walls, and, especially in Langsdorffia, by the presence of collapsed matrix cells between the bundle sheath and tuber matrix. Sheath-conducting tissues consist of densely cytoplasmic transfer cells and small sieve tube members; in Langsdorffia, tracheary elements are also present. These sheath bundles connect with vascular bundles of the tuber matrix. Direct host/parasite contact only occurs by means of parasite transfer cells in the composite bundles. There is no xylem-xylem contact at the host/parasite interface. Abundance of parasite transfer cells suggests that they play an important role in nutrient absorption and translocation.     

Evidence from NMR interaction studies challenges the hypothesis of direct lipid transfer from L‐FABP to malaria sporozoite protein UIS3

UIS3 is a malaria parasite protein essential for liver stage development of Plasmodium species, presumably localized to the membrane of the parasitophorous vacuole formed in infected cells. It has been recently proposed that the soluble domain of UIS3 interacts with the host liver fatty acid binding protein (L‐FABP), providing the parasite with a pathway for importing exogenous lipids required for its rapid growth. This finding may suggest novel strategies for arresting parasite development. In this study, we have investigated the interaction between human L‐FABP and the soluble domain of Plasmodium falciparum UIS3 by NMR spectroscopy. The amino acid residue‐specific analysis of ¹H,¹⁵N‐2D NMR spectra excluded the occurrence of a direct interaction between L‐FABP (in its unbound and oleate‐loaded forms) and Pf‐UIS3. Furthermore, the spectrum of Pf‐UIS3 was unchanged when oleate or phospholipids were added. The present investigation entails a reformulation of the current model of host‐pathogen lipid transfer, possibly redirecting research for early intervention against malaria.     

Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii

Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite''s carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.     

Thy-1 is an in vivo and in vitro marker of liver myofibroblasts

Thy-1, a glycophosphatidylinositol-linked glycoprotein of the outer membrane leaflet, has been described in myofibroblasts of several organs. Previous studies have shown that, in fetal liver, Thy-1 is expressed in a subpopulation of ductular/progenitor cells. The aim of this study has been to investigate whether the liver myofibroblasts belong to the Thy-1-positive subpopulation of the adult liver. The expression of Thy-1 has been studied in normal rat liver, in the rat liver regeneration model following 2-acetylaminofluorene treatment and partial hepatectomy (AAF/PH), and in isolated rat liver cells, at the mRNA and protein levels. In normal rat liver, Thy-1 is detected in sparse cells of the periportal area, whereas 7 days after PH in the AAF/PH model, a marked increase of the number of Thy-1-positive cells is detectable by immunohistochemistry. Comparative immunohistochemical analysis has revealed the co-localization of Thy-1 and smooth muscle actin, but not of Thy-1 and cytokeratin-19, both in normal rat liver and in the AAF/PH model. Investigation of isolated rat liver cell populations has confirmed that liver myofibroblasts are Thy-1-positive cells, whereas hepatocytes, hepatic stellate cells, and liver macrophages are not. Thy-1 is the first cell surface marker for identifying liver myofibroblasts in vivo and in vitro. Jozsef Dudas and Tümen Mansuroglu contributed equally to this study. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 402, projects C6, D3, D4).     

The fine structure and cytology of the association between the parasitic red algaHolmsella australis and its red algal hostGracilaria furcellata

Summary Holmsella australis Noble andKraft ms. is a colourless red algal parasite, forming whitish pustules on its photosynthetic red algal host,Gracilaria furcellata Harvey. In the infected region, host cortical tissue continues to grow and enclose the expanding pustule. Filaments of both host and parasite grow apically, the cells being connected by primary pit connections (PCs). Secondary PCs form between cells of the same species, and in addition,H. australis initiates the formation of secondary PCs with cells ofG. furcellata. All three types of secondary PC are morphologically distinct. In hostparasite PCs the surface adjoining the host cell is similar in structure to a host-host PC, while that adjoining the parasite cell has the structure of a parasite-parasite PC. The plasma membrane is continuous between the cells of the unrelated host and parasite. In addition, a cap membrane is typically produced only on the host surface, though occasionally the parasite side is enclosed by a cap membrane as well. Cap membranes are absent from parasite-parasite PCs (making them intracellular), while host-host PCs are typically extracellular, both cells producing cap membranes. The presence or absence of a cap membrane in certain positions appears to vary, and suggests that cells may be able to regulate its presence. Since transport of nutrients would be expected to occur from host to parasite cells, and between parasite cells, the morphological evidence presented here suggests the PCs may be the pathway.     

The polypeptide components of the parasporal fibres of Pasteuria penetrans

A variety of treatments were tested for their ability to solubilize the parasporal fibres from Pasteuria penetrans, a parasite of some plant–parasitic nematodes. Selective solubilization of the parasporal fibres resulted from some of the extraction procedures tested. Subsequent acrylamide gel electrophoresis and Western blotting of the resolved polypeptides, using polyclonal sera against the spores, disclosed up to 15 distinct bands, ranging in size from 12 to 195 kDa. An N-terminal amino acid sequence was obtained from a 50 kDa polypeptide and an oligonucleotide primer deduced from it. A whole cell, fluorescent, primed in situ labelling (PRINS) technique was adapted to be applicable to spores of P. penetrans and P. ramosa, a parasite of water fleas. Positive responses were obtained using the parasporal fibre primer on spores of the former but not of the latter organism, implying that this 50 kDa polypeptide is produced by P. penetrans but not by P. ramosa.     

Identification of receptors in the liver that mediate endocytosis of circulating tissue kallikreins

The liver plays an important role in the clearance, by receptor-mediated endocytosis, of circulating glycoproteins. It has been demonstrated that tissue kallikreins, which are acid glycoproteins, circulate in plasma, where they are poorly inhibited by plasma proteins. We have shown that the liver is the main organ that clears tissue kallikreins from the circulation. We now report the identification of receptors involved in this clearance. Using a perfused rat-liver system, and as models, pig pancreatic (PPK) and horse urinary (HoUK) kallikreins, we have found that: (a) the binding of PPK to the perfused liver was inhibited by 50 mM methyl α-d-mannoside and 20 μM mannan, was partially inhibited by 50 mM mannose and was unaffected by 1.5 μM asialofetuin; (b) binding of HoUK to the perfused liver was inhibited by 1.5 μM asialofetuin, 50 mM galactose and 50 mM lactose and was unaffected by 50 mM mannose; (c) the clearance rate of both kallikreins followed the equation y = a·x^b; (d) their binding was Ca²⁺-dependent and their clearance was inhibited by 3 mM chloroquine and 10 mM methylamine. Using isolated liver cells and tritiated HoUK, we calculated that 500 000 receptors/cell were present and the Scatchard plot showed that there were two apparent affinity constants: 0.24·10⁹ 1/M)(high-affinity) and 0.3·10⁸ 1/M (low-affinity). These results show that PPK is recognized by a liver mannose receptor and HoUK by the galactose receptor. The liver uptake of native and circulating tissue kallikreins thus emerges as a mechanism by which their levels in plasma are regulated.     

Decellularized Wharton's jelly extracellular matrix as a promising scaffold for promoting hepatic differentiation of human induced pluripotent stem cells

Liver tissue engineering as a therapeutic option for restoring of damaged liver function has a special focus on using native decellularized liver matrix, but there are limitations such as the shortage of liver donor. Therefore, an appropriate alternative scaffold is needed to circumvent the donor shortage. This study was designed to evaluate hepatic differentiation of human induced pluripotent stem cells (hiPSCs) in decellularized Wharton's jelly (WJ) matrix as an alternative for native liver matrix. WJ matrices were treated with a series of detergents for decellularization. Then hiPSCs were seeded into decellularized WJ scaffold (DWJS) for hepatic differentiation by a defined induction protocol. The DNA quantitative assay and histological evaluation showed that cellular and nuclear materials were efficiently removed and the composition of extracellular matrix was maintained. In DWJS, hiPSCs-derived hepatocyte-like cells (hiPSCs-Heps) efficiently entered into the differentiation phase (G1) and gradually took a polygonal shape, a typical shape of hepatocytes. The expression of hepatic-associated genes (albumin, TAT, Cytokeratin19, and Cyp7A1), albumin and urea secretion in hiPSCs-Heps cultured into DWJS was significantly higher than those cultured in the culture plates (2D). Altogether, our results suggest that DWJS could provide a proper microenvironment that efficiently promotes hepatic differentiation of hiPSCs.