Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is followed by dramatic modifications of erythrocytes properties, including de novo formation of new membrane systems. Lipid transfer proteins from both the parasite and the host cell are most likely an important part of those membrane remodeling processes. Using bioinformatics and in silico structural analysis, we have identified five P. falciparum potential lipid transfer proteins containing cellular retinaldehyde binding - triple functional domain (CRAL-TRIO). Two of these proteins, C6KTD4, encoded by the PF3D7_0629900 gene and Q8II87, encoded by the PF3D7_1127600 gene, were studied in more detail. In vitro lipid transfer assays using recombinant C6KTD4 and Q8II87 confirmed that these proteins are indeed bona fide lipid transfer proteins. C6KTD4 transfers sterols, phosphatidylinositol 4,5 bisphosphate, and, to some degree, also phosphatidylcholine between two membrane compartments. Q8II87 possesses phosphatidylserine transfer activity in vitro. In the yeast model, the expression of P. falciparumQ8II87 protein partially complements the absence of Sec14p and its closest homologue, Sfh1p. C6KTD4 protein can substitute for the collective essential function of oxysterol-binding related proteins. According to published whole genome studies in P. falciparum, absence of C6KTD4 and Q8II87 proteins has severe consequences for parasite viability. Therefore, CRAL-TRIO lipid transfer proteins of P. falciparum are potential targets of novel antimalarials, in search for which the yeast model expressing these proteins could be a valuable tool.

• Klíčová slova
• Lipid transfer proteins, Malaria, Phospholipids, Plasmodium falciparum, Saccharomyces cerevisiae, Sterols,
• MeSH
• erytrocyty parazitologie   metabolismus   MeSH
• lidé MeSH
• Plasmodium falciparum * genetika   metabolismus   MeSH
• protozoální proteiny * metabolismus   genetika   chemie   MeSH
• transportní proteiny * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lipid transfer protein MeSH Prohlížeč
• protozoální proteiny * MeSH
• transportní proteiny * MeSH

In a febrile patient admitted to the Adult Emergency Department, the haematology analyser detected the presence of erythrocytes infected with plasmodia. The finding was confirmed by thin smear and thick drop microscopy. A 43-year-old male patient was admitted to the Emergency Department with fever, vomiting, diarrhoea and pain in the upper abdomen. He reported a history of travel to Ethiopia and a short stay in Tanzania. The Sysmex XN series haematology analyser showed the activation of the iRBC flag signalling the presence of Plasmodium-infected erythrocytes. Thin smear and thick drop microscopy confirmed the presence of malaria plasmodia in erythrocytes.

• Klíčová slova
• Malaria, Plasmodium, Sysmex XN series, erythrocyte, haematology analyser, red cells,
• MeSH
• dospělí MeSH
• erytrocyty parazitologie   MeSH
• lidé MeSH
• malárie diagnóza   MeSH
• nemocnice univerzitní MeSH
• urgentní služby nemocnice * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Geografické názvy
• Etiopie MeSH
• Tanzanie MeSH

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr-WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens, providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions.

• Klíčová slova
• 6-cys-e gene knockout, Babesia divergens, GFP-expression, bidirectional promoter, erythrocytes pre-loading, gene targeting, transfection system,
• MeSH
• Babesia * genetika   MeSH
• babezióza * parazitologie   MeSH
• erytrocyty parazitologie   MeSH
• genový targeting MeSH
• lidé MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Phosphoinositide lipids play key roles in a variety of processes in eukaryotic cells, but our understanding of their functions in the malaria parasite Plasmodium falciparum is still very much limited. To gain a deeper comprehension of the roles of phosphoinositides in this important pathogen, we attempted gene inactivation for 24 putative effectors of phosphoinositide metabolism. Our results reveal that 79% of the candidates are refractory to genetic deletion and are therefore potentially essential for parasite growth. Inactivation of the gene coding for a Plasmodium-specific putative phosphoinositide-binding protein, which we named PfPX1, results in a severe growth defect. We show that PfPX1 likely binds phosphatidylinositol-3-phosphate and that it localizes to the membrane of the digestive vacuole of the parasite and to vesicles filled with host cell cytosol and labeled with endocytic markers. Critically, we provide evidence that it is important in the trafficking pathway of hemoglobin from the host erythrocyte to the digestive vacuole. Finally, inactivation of PfPX1 renders parasites resistant to artemisinin, the frontline antimalarial drug. Globally, the minimal redundancy in the putative phosphoinositide proteins uncovered in our work supports that targeting this pathway has potential for antimalarial drug development. Moreover, our identification of a phosphoinositide-binding protein critical for the trafficking of hemoglobin provides key insight into this essential process. IMPORTANCE Malaria represents an enormous burden for a significant proportion of humanity, and the lack of vaccines and problems with drug resistance to all antimalarials demonstrate the need to develop new therapeutics. Inhibitors of phosphoinositide metabolism are currently being developed as antimalarials but our understanding of this biological pathway is incomplete. The malaria parasite lives inside human red blood cells where it imports hemoglobin to cover some of its nutritional needs. In this work, we have identified a phosphoinositide-binding protein that is important for the transport of hemoglobin in the parasite. Inactivation of this protein decreases the ability of the parasite to proliferate. Our results have therefore identified a potential new target for antimalarial development.

• Klíčová slova
• hemoglobin, knockout, malaria, phosphoinositides, vacuoles, vesicular trafficking,
• MeSH
• antimalarika * farmakologie   MeSH
• erytrocyty parazitologie   MeSH
• fosfatidylinositoly metabolismus   MeSH
• hemoglobiny metabolismus   MeSH
• lidé MeSH
• malárie MeSH
• paraziti metabolismus   MeSH
• Plasmodium falciparum * genetika   MeSH
• protozoální proteiny * genetika   MeSH
• transportní proteiny metabolismus   MeSH
• tropická malárie * genetika   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antimalarika * MeSH
• fosfatidylinositoly MeSH
• hemoglobiny MeSH
• protozoální proteiny * MeSH
• transportní proteiny MeSH

Parasites of the Plasmodium genus are unable to produce purine nucleotides de novo and depend completely on the salvage pathway. This fact makes plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] a valuable target for development of antimalarial agents. A series of nucleotide analogues was designed, synthesized and evaluated as potential inhibitors of Plasmodium falciparum HGXPRT, P. vivax HGPRT and human HGPRT. These novel nucleoside phosphonates have a pyrrolidine, piperidine or piperazine ring incorporated into the linker connecting the purine base to a phosphonate group(s) and exhibited a broad range of Ki values between 0.15 and 72 μM. The corresponding phosphoramidate prodrugs, able to cross cell membranes, have been synthesized and evaluated in a P. falciparum infected human erythrocyte assay. Of the eight prodrugs evaluated seven exhibited in vitro antimalarial activity with IC50 values within the range of 2.5-12.1 μM. The bis-phosphoramidate prodrug 13a with a mean (SD) IC50 of 2.5 ± 0.7 μM against the chloroquine-resistant P. falciparum W2 strain exhibited low cytotoxicity in the human hepatocellular liver carcinoma (HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of 100 μM suggesting good selectivity for further structure-activity relationship investigations.

• Klíčová slova
• HG(X)PRT, Hypoxanthine-guanine-(xanthine) phosphoribosyltransferase, Nucleoside phosphonates, Phosphoroamidate prodrug, Plasmodium falciparum, Plasmodium vivax,
• MeSH
• antimalarika chemická syntéza   metabolismus   farmakologie   MeSH
• buněčné linie MeSH
• erytrocyty cytologie   metabolismus   parazitologie   MeSH
• inhibitory enzymů chemie   metabolismus   MeSH
• léková rezistence účinky léků   MeSH
• lidé MeSH
• nukleotidy chemie   metabolismus   MeSH
• pentosyltransferasy antagonisté a inhibitory   metabolismus   MeSH
• piperazin chemie   MeSH
• piperidiny chemie   MeSH
• Plasmodium falciparum účinky léků   enzymologie   MeSH
• Plasmodium vivax enzymologie   MeSH
• preklinické hodnocení léčiv MeSH
• prekurzory léčiv chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• protozoální proteiny antagonisté a inhibitory   metabolismus   MeSH
• pyrrolidiny chemie   MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antimalarika MeSH
• hypoxanthine-guanine-xanthine phosphoribosyltransferase MeSH Prohlížeč
• inhibitory enzymů MeSH
• nukleotidy MeSH
• pentosyltransferasy MeSH
• piperazin MeSH
• piperidiny MeSH
• prekurzory léčiv MeSH
• protozoální proteiny MeSH
• pyrrolidine MeSH Prohlížeč
• pyrrolidiny MeSH

AIMS: As the most abundant cell population in the blood, erythrocytes represent an attractive source of nutrients and a protective niche to a number of pathogens. Previously, we observed the attachment of the myxozoan parasite Sphaerospora molnari to erythrocytes of its host, common carp (Cyprinus carpio), raising a number of questions about the nature of this interaction. METHODS AND RESULTS: We elucidated the impact of S molnari on the number of erythrocytes in healthy and immunocompromised fish, over a period of 6 weeks. While we observed only a mild decrease in RBC numbers in healthy individuals, we witnessed gradual and finally severe haemolytic anaemia in immunosuppressed fish. Accompanying this overt loss was increased erythropoiesis as represented by an increase of erythroblasts in the blood. In vitro, we demonstrated the uptake of host proteins from CFSE-labelled erythrocytes, ultimately inducing death of host RBCs, likely for nutrient gain of the parasite. Nevertheless, the results do not exclude a possible role of erythrocyte-derived proteins in immune evasion. CONCLUSION: Overall, the obtained data provide first evidence for the previously unknown appetite of myxozoan parasites for host erythrocytes and create an important framework for future investigations into the molecular mechanisms underlining this interaction.

• Klíčová slova
• Myxozoa, common carp, erythrocytes, flow cytometry, haemolytic anaemia, hematophagy,
• MeSH
• erytrocyty parazitologie   MeSH
• erytropoéza fyziologie   MeSH
• fylogeneze MeSH
• hemolytické anemie parazitologie   MeSH
• kapři parazitologie   MeSH
• Myxozoa fyziologie   MeSH
• nemoci ryb parazitologie   MeSH
• stravovací zvyklosti fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes, it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of nonoptimal peptide substrates. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.

• Klíčová slova
• dipeptidyl aminopeptidase, malaria, positional scanning, proteases, specificity,
• MeSH
• aminokyseliny chemie   MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy metabolismus   MeSH
• erytrocyty účinky léků   metabolismus   parazitologie   MeSH
• inhibitory proteas farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• peptidové fragmenty metabolismus   MeSH
• Plasmodium falciparum účinky léků   růst a vývoj   metabolismus   MeSH
• substrátová specifita MeSH
• tropická malárie farmakoterapie   metabolismus   parazitologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• dipeptidyl peptidase III MeSH Prohlížeč
• dipeptidylpeptidasy a tripeptidylpeptidasy MeSH
• inhibitory proteas MeSH
• peptidové fragmenty MeSH

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures.

• MeSH
• erytrocyty metabolismus   parazitologie   MeSH
• hemoproteiny analýza   MeSH
• konfokální mikroskopie metody   MeSH
• mikroskopie atomárních sil metody   MeSH
• Plasmodium falciparum chemie   růst a vývoj   metabolismus   ultrastruktura   MeSH
• Ramanova spektroskopie metody   MeSH
• spektrofotometrie infračervená metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemoproteiny MeSH
• hemozoin MeSH Prohlížeč

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of PfEMP1s and inhibits ICAM-1 binding. The 24E9 mouse mAb was raised against PFD1235w DBLβ3_D4, a domain from the group A PfEMP1s associated with severe malaria. 24E9 recognizes native PfEMP1 expressed on the IE surface and shows cross-reactivity with and cross-inhibition of the ICAM-1 binding capacity of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLβ3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides that are solvent protected in the complex. When mapped onto a homology model of DBLβ3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLβ3_D4. Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLβ3_D4::24E9 Fab complex derived from small-angle x-ray scattering. The convex surface of DBLβ3_D4 has previously been shown to contain the ICAM-1 binding site of DBLβ domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface. Conserved epitopes, such as those targeted by 24E9, are promising candidates for the inclusion in a vaccine interfering with ICAM-1-specific adhesion of group A PfEMP1 expressed by P. falciparum IE during severe malaria.

• MeSH
• antigeny protozoální imunologie   MeSH
• buněčná adheze MeSH
• cévní endotel metabolismus   parazitologie   MeSH
• epitopy imunologie   MeSH
• erytrocytární membrána imunologie   MeSH
• erytrocyty parazitologie   MeSH
• hybridomy MeSH
• kultivované buňky MeSH
• mezibuněčná adhezivní molekula-1 imunologie   MeSH
• molekulární sekvence - údaje MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• Plasmodium falciparum imunologie   MeSH
• protilátky protozoální imunologie   MeSH
• protozoální proteiny imunologie   MeSH
• terciární struktura proteinů MeSH
• tropická malárie imunologie   parazitologie   MeSH
• vazebná místa protilátek imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny protozoální MeSH
• epitopy MeSH
• erythrocyte membrane protein 1, Plasmodium falciparum MeSH Prohlížeč
• mezibuněčná adhezivní molekula-1 MeSH
• monoklonální protilátky MeSH
• protilátky protozoální MeSH
• protozoální proteiny MeSH

Two experimental trials were performed to elucidate the role of rodents in the life cycle of Hepatozoon species using snakes as intermediate hosts. In one trial, two ball pythons, Python regius Shaw, 1802 were force fed livers of laboratory mice previously inoculated with sporocysts of Hepatozoon ayorgbor Sloboda, Kamler, Bulantová, Votýpka et Modrý, 2007. Transmission was successful in these experimentally infected snakes as evidenced by the appearance of intraerythrocytic gamonts, which persisted until the end of trial, 12 months after inoculation. Developmental stages of haemogregarines were not observed in histological sections from mice. In another experimental trial, a presence of haemogregarine DNA in mice inoculated with H. ayorgbor was demonstrated by PCR in the liver, lungs and spleen.

• MeSH
• Apicomplexa izolace a purifikace   MeSH
• Boidae parazitologie   MeSH
• erytrocyty parazitologie   MeSH
• hlodavci parazitologie   MeSH
• infekce přenášené vektorem * MeSH
• játra parazitologie   MeSH
• myši MeSH
• plíce parazitologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• protozoální DNA izolace a purifikace   MeSH
• protozoální infekce zvířat přenos   MeSH
• slezina parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální DNA MeSH