Protein Binder Toolbox for Studies of Solute Carrier Transporters.

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase.

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement-a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound-and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

Xenosurveillance proof-of-principle: Detection of Toxoplasma gondii and SARS-CoV-2 antibodies in mosquito blood meals by (pan)-specific ELISAs.

Blood-fed insects can be used to analyse the host blood for circulating vertebrate pathogens or antibodies directed against them. We tested whether naturally acquired antibodies in different host species can be detected by host-specific and pan-specific ELISAs in mosquito blood meals. Cat- and alpaca-specific ELISAs could detect antibodies against Toxoplasma gondii or SARS-CoV-2 in blood meals of Aedes japonicus for 48 and at least 24 h, respectively. In the pan-specific ELISA, a conjugated protein A/G and anti-IgY were used to detect antibodies of mammalian and bird hosts. Thus, Toxoplasma antibodies could be detected in mosquitoes fed on blood from humans, chicken, pig, and sheep up to 72 h after the blood meal. The results, however, demonstrated differences in sensitivities between different host species, and the assay requires further evaluation. Xenosurveillance with antibody detection in mosquito blood meals can be an additional surveillance tool that would especially be helpful when it is difficult to sample the potential animal reservoirs.

Serine administration as a novel prophylactic approach to reduce the severity of acute pancreatitis during diabetes in mice.

AIMS/HYPOTHESIS: Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting. METHODS: We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro. RESULTS: We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis. CONCLUSIONS/INTERPRETATION: Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.

The extracellular gate shapes the energy profile of an ABC exporter.

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

Highly modified and immunoactive N-glycans of the canine heartworm.

The canine heartworm (Dirofilaria immitis) is a mosquito-borne parasitic nematode whose range is extending due to climate change. In a four-dimensional analysis involving HPLC, MALDI-TOF-MS and MS/MS in combination with chemical and enzymatic digestions, we here reveal an N-glycome of unprecedented complexity. We detect N-glycans of up to 7000 Da, which contain long fucosylated HexNAc-based repeats, as well as glucuronylated structures. While some modifications including LacdiNAc, chitobiose, α1,3-fucose and phosphorylcholine are familiar, anionic N-glycans have previously not been reported in nematodes. Glycan array data show that the neutral glycans are preferentially recognised by IgM in dog sera or by mannose binding lectin when antennal fucose and phosphorylcholine residues are removed; this pattern of reactivity is reversed for mammalian C-reactive protein, which can in turn be bound by the complement component C1q. Thereby, the N-glycans of D. immitis contain features which may either mediate immunomodulation of the host or confer the ability to avoid immune surveillance.

An ELISA for the early diagnosis of acute canine babesiosis detecting circulating antigen of large Babesia spp.

Babesia canis is the predominant Babesia species in dogs in Europe and is responsible for a severe and fatal disease. An increase in global pet tourism and a widening of the geographic distribution of the tick vector has led to the emergence of infections in areas where previously only imported cases have been reported. Due to the potential for rapid and serious disease progression, direct parasite detection by stained blood smears and light microscopy or DNA-based methods have traditionally been used for the diagnosis of acute infections. This study describes the production of a murine monoclonal antibody ('mAb BcFIII 7/1/2') that reacts to a 65kDa corpuscular epitope present in B. canis-infected erythrocytes and can be used in an ELISA to detect circulating Babesia antigen during acute infections. The sensitivity of the ELISA was 100% (95%CI: 84.5-100) as determined using blood lysate samples from 27 dogs with acute B. canis infections. Sensitivity was reduced to 53.8% in 13 patent Babesia vogeli infections (95%CI: 26.1-79.6) based on the current test design using convalescent serum from a B. canis-infected dog. The specificity was determined to be 86.4% (95%CI: 64-96.4) using 22 samples from healthy canine blood donors. In the course of acute B. canis infections, the ELISA showed a positive result at the same time as a positive PCR result was recorded. This was 24-48h before parasites could be detected by light microscopy. Convalescent samples collected from 6 B. canis-infected dogs at least 14days post treatment resulted in negative ELISA reactions. The hyper-acute to acute phase of a B. canis infection represents an emergency situation with high mortality. To increase the chances of survival, a fast and accurate diagnosis and immediate treatment is required. The current study demonstrates the opportunity of an early and specific detection of acute infections by an AgELISA that is potentially translatable to a rapid diagnostic test design.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

Inhibition of Haemonchus contortus larval development by fungal lectins.

BACKGROUND: Lectins are carbohydrate-binding proteins that are involved in fundamental intra- and extracellular biological processes. They occur ubiquitously in nature and are especially abundant in plants and fungi. It has been well established that certain higher fungi produce lectins in their fruiting bodies and/or sclerotia as a part of their natural resistance against free-living fungivorous nematodes and other pests. Despite relatively high diversity of the glycan structures in nature, many of the glycans targeted by fungal lectins are conserved among organisms of the same taxon and sometimes even among different taxa. Such conservation of glycans between free-living and parasitic nematodes is providing us with a useful tool for discovery of novel chemotherapeutic and vaccine targets. In our study, a subset of fungal lectins emanating from toxicity screens on Caenorhabditis elegans was tested for their potential to inhibit larval development of Haemonchus contortus. METHODS: The effect of Coprinopsis cinerea lectins - CCL2, CGL2, CGL3; Aleuria aurantia lectin - AAL; Marasmius oreades agglutinin - MOA; and Laccaria bicolor lectin - Lb-Tec2, on cultivated Haemonchus contortus larval stages was investigated using a larval development test (LDT). To validate the results of the toxicity assay and determine lectin binding capacity to the nematode digestive tract, biotinylated versions of lectins were fed to pre-infective larval stages of H. contortus and visualized by fluorescent microscopy. Lectin histochemistry on fixed adult worms was performed to investigate the presence and localisation of lectin binding sites in the disease-relevant developmental stage. RESULTS: Using an improved larval development test we found that four of the six tested lectins: AAL, CCL2, MOA and CGL2, exhibited a dose-dependent toxicity in LDT, as measured by the number of larvae developing to the L3 stage. In the case of AAL, CGL2 and MOA lectin, doses as low as 5 µg/ml caused >95 % inhibition of larval development while 40 µg/ml were needed to achieve the same inhibition by CCL2 lectin. MOA was the only lectin tested that caused larval death while other toxic lectins had larvistatic effect manifesting as L1 growth arrest. Using lectin histochemistry we demonstrate that of all lectins tested, only the four toxic ones displayed binding to the larvae's gut and likewise were found to interact with glycans localized to the gastrodermal tissue of adults. CONCLUSION: The results of our study suggest a correlation between the presence of target glycans of lectins in the digestive tract and the lectin-mediated toxicity in Haemonchus contortus. We demonstrate that binding to the structurally conserved glycan structures found in H. contortus gastrodermal tissue by the set of fungal lectins has detrimental effect on larval development. Some of these glycan structures might represent antigens which are not exposed to the host immune system (hidden antigens) and thus have a potential for vaccine or drug development. Nematotoxic fungal lectins prove to be a useful tool to identify such targets in parasitic nematodes.

Prolyl Oligopeptidase from the Blood Fluke Schistosoma mansoni: From Functional Analysis to Anti-schistosomal Inhibitors.

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. CONCLUSIONS/SIGNIFICANCE: We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs.

RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening.

BACKGROUND: The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. METHODOLOGY/PRINCIPAL FINDINGS: We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite. CONCLUSIONS: Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.

Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes.

A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote.

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of ;naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.

Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells.

A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents.

Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia.

During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.

Polymerase chain reaction for detection of patent infections of Echinococcus granulosus ("sheep strain") in naturally infected dogs.

Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus ("sheep strain") was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a Echinococcus multilocularis -specific PCR revealing one mixed infection. Egg-containing faeces from two dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental samples.