Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg) of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract.
Ascaris suum is an intestinal parasitic nematode of pigs that is very closely related to Ascaris lumbricoides, a major intestinal parasitic nematode of humans that infects more than one billion people worldwide. Because of reduced efficacy and the threat of resistance to the current small set of approved drugs to treat Ascaris infections, new treatments are needed. Here we test against A. suum infections the effectiveness of Cry5B, a nematode-killing protein made by the natural soil bacterium Bacillus thuringiensis and representing a promising new class of anthelmintics. We demonstrate for the first time that A. suum possesses the receptors to bind Cry5B and that Cry5B can kill A. suum larvae and adults in culture. Most importantly, we demonstrate that oral administration of Cry5B to pigs infected with A. suum larvae results in a near complete elimination of the infection. Given the similarities between A. suum and A. lumbricoides and the similarity between the pig and human gastrointestinal tracts, our data indicate that Cry5B has excellent potential to treat Ascaris infections in veterinary animals and in humans.
A hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase involved in chlorogenic acid biosynthesis in C. canephora was crystallized using the vapour-diffusion method. A diffraction data set was collected to 3.0 Å resolution on the microfocus beamline (ID23-2) at ESRF and a structure solution was obtained using molecular replacement.
Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, including Coffea canephora (robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT from C. canephora in Escherichia coli as well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293 K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space group P42212 with unit-cell parameters a = b = 116.1, c = 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily.
Coffea canephora; phenylpropanoid-biosynthesis pathway; chlorogenic acids; plant acyl-CoA-dependent acyltransferase superfamily; hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase; molecular replacement
Biliverdin reductase (BVR) from Synechocystis sp. PCC6803 and its selenomethionine derivative were overexpressed and purified. X-ray diffraction data from an SeMet BVR microcrystal were collected to 3.0 Å resolution on microfocus beamline BL32XU at SPring-8.
Biliverdin reductase (BVR) catalyzes the conversion of biliverdin IX α to bilirubin IX α with concomitant oxidation of an NADH or NADPH cofactor. This enzyme also binds DNA and enhances the transcription of specific genes. Recombinant cyanobacterial BVR was overexpressed in Escherichia coli, purified and crystallized. A native data set was collected to 2.34 Å resolution on beamline BL38B1 at SPring-8. An SeMet data set was collected from a microcrystal (300 × 10 × 10 µm) on the RIKEN targeted protein beamline BL32XU and diffraction spots were obtained to 3.0 Å resolution. The native BVR crystal belonged to space group P212121, with unit-cell parameters a = 58.8, b = 88.4, c = 132.6 Å. Assuming that two molecules are present in the asymmetric unit, V
M (the Matthews coefficient) was calculated to be 2.37 Å3 Da−1 and the solvent content was estimated to be 48.1%. The structure of cyanobacterial BVR may provide insights into the mechanisms of its enzymatic and physiological functions.
bilirubin; biliverdin; microcrystals; microfocus beamline
Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.
The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.
This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.
Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris, and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structure.
The room-temperature structure of lysozyme is determined using 40000 individual diffraction patterns from micro-crystals flowing in liquid suspension across a synchrotron microfocus beamline.
A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.
serial crystallography; room-temperature protein crystallography; radiation damage; CrystFEL; microfocus beamline
The key features of the functionality facilitating proprietary use of the ESRF’s structural biology beamlines are described, as are the major advantages, in terms of beamline evolution, of the interaction of the ESRF with the pharmaceutical industry.
The ESRF has worked with, and provided services for, the pharmaceutical industry since the construction of its first protein crystallography beamline in the mid-1990s. In more recent times, industrial clients have benefited from a portfolio of beamlines which offer a wide range of functionality and beam characteristics, including tunability, microfocus and micro-aperture. Included in this portfolio is a small-angle X-ray scattering beamline dedicated to the study of biological molecules in solution. The high demands on throughput and efficiency made by the ESRF’s industrial clients have been a major driving force in the evolution of the ESRF’s macromolecular crystallography resources, which now include remote access, the automation of crystal screening and data collection, and a beamline database allowing sample tracking, experiment reporting and real-time at-a-distance monitoring of experiments. This paper describes the key features of the functionality put in place on the ESRF structural biology beamlines and outlines the major advantages of the interaction of the ESRF with the pharmaceutical industry.
synchrotron MX beamlines; proprietary access; service data collection; automation
The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase.
A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB_REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.
carboxypeptidases; metalloproteins; refinement; specificity; zinc enzymes
A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines. This tool provides the possibility of automating complex experiments without the need for expertise in instrumentation control and programming, but rather by accessing defined beamline services.
The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal reorientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE.
workflows; automation; data processing; macromolecular crystallography; experimental protocols; characterization; reorientation; radiation damage
Optical trapping has successfully been applied to select and mount microcrystals for subsequent X-ray diffraction experiments.
X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25 µm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.
laser tweezers; optical trapping; microcrystals; crystal manipulation; sample holders
Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism.
In the present study, we constructed a cDNA library enriched for molecules of the infective third-stage larva (L3) of Ascaris suum, the common roundworm of pigs. Using the method of suppressive-subtractive hybridization (SSH), we explored transcription of a subset of molecules by microarray analysis and conducted bioinformatic analyses to characterize these molecules, map them to biochemical pathways, and predict genetic interactions based on comparisons with Caenorhabditis elegans and/or other organisms. The results provide interesting insights into early molecular processes in A. suum. Approximately 60% of the L3-enriched molecules discovered had homologues in C. elegans. Probabilistic analyses suggested that a complex genetic network regulates or controls larval growth and development in A. suum L3s, some of which might be involved in or regulate the switch from the free-living to the parasitic stage. Functional studies of these molecules to elucidate developmental processes in Ascaris could assist in identifying new targets for intervention.
A retrospective analysis of radiation damage behaviour in a statistically significant number of real-life datasets is presented, in order to gauge the importance of the complications not yet measured or rigorously evaluated in current experiments, and the challenges that remain before radiation damage can be considered a problem solved in practice.
The radiation damage behaviour in 43 datasets of 34 different proteins collected over a year was examined, in order to gauge the reliability of decay metrics in practical situations, and to assess how these datasets, optimized only empirically for decay, would have benefited from the precise and automatic prediction of decay now possible with the programs RADDOSE [Murray, Garman & Ravelli (2004 ▶). J. Appl. Cryst.
37, 513–522] and BEST [Bourenkov & Popov (2010 ▶). Acta Cryst. D66, 409–419]. The results indicate that in routine practice the diffraction experiment is not yet characterized well enough to support such precise predictions, as these depend fundamentally on three interrelated variables which cannot yet be determined robustly and practically: the flux density distribution of the beam; the exact crystal volume; the sensitivity of the crystal to dose. The former two are not satisfactorily approximated from typical beamline information such as nominal beam size and transmission, or two-dimensional images of the beam and crystal; the discrepancies are particularly marked when using microfocus beams (<20 µm). Empirically monitoring decay with the dataset scaling B factor (Bourenkov & Popov, 2010 ▶) appears more robust but is complicated by anisotropic and/or low-resolution diffraction. These observations serve to delineate the challenges, scientific and logistic, that remain to be addressed if tools for managing radiation damage in practical data collection are to be conveniently robust enough to be useful in real time.
radiation damage; data collection; strategy; beamline software; datasets
IREM-1 (immune receptor expressed on myeloid cells 1) is an inhibitory receptor of the immunoglobulin superfamily involved in the functional regulation of myeloid cells. It contains an extracellular region comprising one IgV-like domain. The extracellular IgV-like domain, believed to be involved in target-cell recognition, has been expressed in E. coli, folded in vitro and crystallized.
IREM-1 is an inhibitory receptor involved in the functional regulation of myeloid cells. The expression, in vitro folding, purification, crystallization and X-ray data collection of the Ig-V like domain of IREM-1 are reported. X-ray data were collected from a microcrystal (300 × 10 × 10 µm) at 100 K and a diffraction pattern was obtained to 2.6 Å resolution on microfocus beamline ID23-2 at the ESRF. The crystal belongs to space group P3121, with unit-cell parameters a = b = 54.23, c = 72.02 Å, α = γ = 90, β = 120°. Assuming the presence of one molecule per asymmetric unit, V
M (the Matthews coefficient) was calculated to be 1.96 Å3 Da−1 and the solvent content was estimated to be 37.27%. Determination of the IREM-1 structure will provide insights into its structural requirements for ligand discrimination and binding.
high-throughput crystallization; ID23-2, inhibitory receptors; myeloid cells; immunoreceptors; refolding
The structure solution of T. brucei cathepsin B from 80 in vivo grown crystals with an average volume of 9 µm3 obtained by serial synchrotron crystallography at a microfocus beamline is reported.
Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.
protein microcrystallography; serial crystallography; in vivo grown microcrystals
The nematode intestine is a major organ responsible for nutrient digestion and absorption; it is also involved in many other processes, such as reproduction, innate immunity, stress responses, and aging. The importance of the intestine as a target for the control of parasitic nematodes has been demonstrated. However, the lack of detailed knowledge on the molecular and cellular functions of the intestine and the level of its conservation across nematodes has impeded breakthroughs in this application.
Methods and Findings
As part of an extensive effort to investigate various transcribed genomes from Ascaris suum and Haemonchus contortus, we generated a large collection of intestinal sequences from parasitic nematodes by identifying 3,121 A. suum and 1,755 H. contortus genes expressed in the adult intestine through the generation of expressed sequence tags. Cross-species comparisons to the intestine of the free-living C. elegans revealed substantial diversification in the adult intestinal transcriptomes among these species, suggesting lineage- or species-specific adaptations during nematode evolution. In contrast, significant conservation of the intestinal gene repertories was also evident, despite the evolutionary distance of ∼350 million years separating them. A group of 241 intestinal protein families (IntFam-241), each containing members from all three species, was identified based on sequence similarities. These conserved proteins accounted for ∼20% of the sampled intestinal transcriptomes from the three nematodes and are proposed to represent conserved core functions in the nematode intestine. Functional characterizations of the IntFam-241 suggested important roles in molecular functions such as protein kinases and proteases, and biological pathways of carbohydrate metabolism, energy metabolism, and translation. Conservation in the core protein families was further explored by extrapolating observable RNA interference phenotypes in C. elegans to their parasitic counterparts.
Our study has provided novel insights into the nematode intestine and lays foundations for further comparative studies on biology, parasitism, and evolution within the phylum Nematoda.
Biological properties of the nematode intestine warrant in-depth investigation, the results of which can be utilized in the control of parasitic nematodes that infect humans, livestock, and plants. Both the importance of intestinal antigens from Haemonchus contortus in immunity and the damage to H. contortus intestine by anthelmintic fenbendazole have highlighted the versatility of the intestine as an emerging target. However, biological information regarding fundamental intestinal cell functions and mechanisms is currently limited. Conserved intestinal genes across nematode pathogens could offer molecular targets for broad parasite control. Furthermore, qualitative and quantitative comparisons on intestinal gene expression among species and lineages can identify basic adaptations relative to a critical selective force, the nutrient acquisition. This study begins to identify intestinal cell characteristics that are conserved across representatives of two clades of nematodes (V and III) and further clarifies diversities that likely reflect species- or lineage-specific adaptations. Results consistent with functional data on digestive enzymes from H. contortus and RNAi in Caenorhabditis elegans, as examples, support the potential for the comparative genomics approach to produce practical applications. This study provides a platform on which extensive investigation of intestinal genes and a more comprehensive understanding of the Nematoda can be gained.
The SIBYLS beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory is a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline. Key features and capabilities are described along with implementation and performance.
The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world’s mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B4C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources.
small-angle X-ray scattering (SAXS); macromolecular crystallography (MX); synchrotron beamlines; SIBYLS
The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.
Past research has demonstrated that the nematode intestine has value for developing new methods of therapy and control of parasitic nematodes, as related to both vaccines and other anthelmintics. Yet, information related to basic intestinal cell biology is very limited. Research progress reported here moves towards the comprehensive identification of proteins (peptidases and others), and hence functions, that are sited on the apical intestinal membrane and within the intestinal lumen of adult female Ascaris suum. These advances provide an unprecedented research model to determine critical functions sited at these locations and to develop approaches to inhibit those functions. Comparative analysis among diverse parasitic species raises expectations that the results from A. suum can be applied to many parasitic nematodes for which similar research is technically impossible to perform.
Strategies employed by parasites to establish infections are poorly understood. The host-parasite interface is maintained through a molecular dialog that, among other roles, protects parasites from host immune responses. Parasite excretory/secretory products (ESP) play major roles in this process. Understanding the biology of protein secretion by parasites and their associated functional processes will enhance our understanding of the roles of ESP in host-parasite interactions.
ESP was collected after culturing 10 adult female Ascaris suum. Perienteric fluid (PE) and uterine fluid (UF) were collected directly from adult females by dissection. Using SDS-PAGE coupled with LC-MS/MS, we identified 175, 308 and 274 proteins in ESP, PE and UF, respectively. Although many proteins were shared among the samples, the protein composition of ESP was distinct from PE and UF, whereas PE and UF were highly similar. The distribution of gene ontology (GO) terms for proteins in ESP, PE and UF supports this claim. Comparison of ESP composition in A. suum, Brugia malayi and Heligmosoides polygyrus showed that proteins found in UF were also secreted by males and by larval stages of other species, suggesting that multiple routes of secretion may be used for homologous proteins. ESP composition of nematodes is both phylogeny- and niche-dependent.
Analysis of the protein composition of A. suum ESP and UF leads to the conclusion that the excretory-secretory apparatus and uterus are separate routes for protein release. Proteins detected in ESP have distinct patterns of biological functions compared to those in UF. PE is likely to serve as the source of the majority of proteins in UF. This analysis expands our knowledge of the biology of protein secretion from nematodes and will inform new studies on the function of secreted proteins in the orchestration of host-parasite interactions.
Ascaris lumbricoides, the most prevalent metazoan parasite of humans, is a public health concern in resource-limited countries. Survival of this parasite in its host is mediated at least in part by parasite materials secreted into the host. Little is known about the composition of these secretions; defining their contents and functions will illuminate host-parasite interactions that lead to parasite establishment. Ascaris suum, a parasite of pigs, was used as a model organism because its genome has been sequenced and it is very closely related to A. lumbricoides. Excretory/secretory products (ESP), uterine fluid (UF) and perienteric fluid (PE) were collected from adult A. suum. Proteins were subjected to LC-MS/MS. ESP proteins (the ‘secretome’) included many also present in UF. Proteins in ESP but not in UF had considerably different characteristics than those in PE or UF, which were similar to each other. We conclude that proteins released from the secretory apparatus have distinct patterns of biological function and that UF proteins are likely derived from PE. Comparing the protein composition of A. suum ESP to ESP from B. malayi and H. polygyrus suggests that the secretome is conserved at the level of both phylogeny and host predilection site.
Over a billion people are infected by Ascaris spp. intestinal parasites. To clarify functional differences among tissues of adult A. suum, we compared gene expression by various tissues of these worms by expression microarray methods.. The A. suum genome was sequenced and assembled to allow generation of microarray elements. Expression of over 40,000 60-mer elements was investigated in a variety of tissues from both male and female adult worms. Nearly 50 percent of the elements for which signal was detected exhibited differential expression among different tissues. The unique profile of transcripts identified for each tissue clarified functional distinctions among tissues, such as chitin binding in the ovary and peptidase activity in the intestines. Interestingly, hundreds of gender-specific elements were characterized in multiple non-reproductive tissues of female or male worms, with most prominence of gender differences in intestinal tissue. A. suum genes from the same family were frequently expressed differently among tissues. Transcript abundance for genes specific to A. suum, by comparison to Caenorhabditis elegans, varied to a greater extent among tissues than for genes conserved between A. suum and C. elegans. Analysis using C. elegans protein interaction data identified functional modules conserved between these two nematodes, resulting in identification of functional predictions of essential subnetworks of protein interactions and how these networks may vary among nematode tissues. A notable finding was very high module similarity between adult reproductive tissues and intestine. Our results provide the most comprehensive assessment of gene expression among tissues of a parasitic nematode to date.
Nematode; parasite; Ascaris suum; tissue; expression
Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism.
Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female.
The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue-specific biological functions in nematodes.
Tissue-specific gene expression provides fundamental information about the biology of diverse cell types within an organism and interactions among tissues within multicellular organisms. However, such studies are experimentally challenging in smaller organisms such as many nematodes species, including the species (Caenorhabditis elegans) that is widely used in biomedical research. Ascaris suum (the large roundworm of swine), however, is of particular interest as a model nematode because it is large enough to allow for the dissection of individual tissues, and equally important because it is closely related to A. lumbricoides, which infects ∼1 billion people worldwide. Here, we build significantly on the previous tissue-specific gene expression research in A. suum by producing the first nematode RNA-seq dataset that spans multiple specific tissues, including three non-reproductive and two reproductive tissues in both male and female A. suum worms. This analysis provides significant details on the biological functions occurring within each of these tissues, which has not been previously explored. It also provides insight into specific gene regulation pathways active in each of the tissues, which have broad applicability across other nematodes, including both non-parasitic and parasitic species.
Beamline ID23-2, the first dedicated and highly automated high-throughput monochromatic macromolecular crystallography microfocus beamline, is described.
The first phase of the ESRF beamline ID23 to be constructed was ID23-1, a tunable MAD-capable beamline which opened to users in early 2004. The second phase of the beamline to be constructed is ID23-2, a monochromatic microfocus beamline dedicated to macromolecular crystallography experiments. Beamline ID23-2 makes use of well characterized optical elements: a single-bounce silicon (111) monochromator and two mirrors in Kirkpatrick–Baez geometry to focus the X-ray beam. A major design goal of the ID23-2 beamline is to provide a reliable, easy-to-use and routine microfocus beam. ID23-2 started operation in November 2005, as the first beamline dedicated to microfocus macromolecular crystallography. The beamline has taken the standard automated ESRF macromolecular crystallography environment (both hardware and software), allowing users of ID23-2 to be rapidly familiar with the microfocus environment. This paper describes the beamline design, the special considerations taken into account given the microfocus beam, and summarizes the results of the first years of the beamline operation.
macromolecular crystallography; automation; microfocus
The ultimate goal of synchrotron data collection is to obtain the best possible data from the best available crystals, and the combination of automation and remote access at Stanford Synchrotron Radiation Lightsource (SSRL) has revolutionized the way in which scientists achieve this goal. This has also seen a change in the way novice crystallographers are trained in the use of the beamlines, and a wide range of remote tools and hands-on workshops are now offered by SSRL to facilitate the education of the next generation of protein crystallographers.
For the past five years, the Structural Molecular Biology group at the Stanford Synchrotron Radiation Lightsource (SSRL) has provided general users of the facility with fully remote access to the macromolecular crystallography beamlines. This was made possible by implementing fully automated beamlines with a flexible control system and an intuitive user interface, and by the development of the robust and efficient Stanford automated mounting robotic sample-changing system. The ability to control a synchrotron beamline remotely from the comfort of the home laboratory has set a new paradigm for the collection of high-quality X-ray diffraction data and has fostered new collaborative research, whereby a number of remote users from different institutions can be connected at the same time to the SSRL beamlines. The use of remote access has revolutionized the way in which scientists interact with synchrotron beamlines and collect diffraction data, and has also triggered a shift in the way crystallography students are introduced to synchrotron data collection and trained in the best methods for collecting high-quality data. SSRL provides expert crystallographic and engineering staff, state-of-the-art crystallography beamlines, and a number of accessible tools to facilitate data collection and in-house remote training, and encourages the use of these facilities for education, training, outreach and collaborative research.
protein crystallography; high-throughput screening; robotics; remote access; crystallographic education and training; outreach
Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES) molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses.
The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4) by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut.
The present proteomic analysis provides important information on the host-parasite interaction and the biology of the migratory stages of A. suum. In particular, the high transcriptional upregulation of glycosyl hydrolases from the L4 stage onwards reveals that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine.
The gastro-intestinal nematodes Ascaris lumbricoides and Ascaris suum are amongst the most prevalent parasites of humans and pigs, respectively. To date, little is known about A. suum excretory-secretory proteins, which are present at the parasite-host interface and likely to play a critical role in the induction and development of the immune response. The aim of this study was to identify the excretory-secretory proteins of the migratory stages of A. suum utilizing LC-MS/MS. In total, 106 proteins were identified, some of which are known as important players in the parasite-host interface. Interestingly, an abundance of glycosyl hydrolases was observed in the ES material of the intestinal L4 stage larvae. By combining the proteomic analysis with in depth genomic, transcriptomic and enzymatic analyses we could show that the glycosyl hydrolase protein family has undergone a massive expansion in A. suum and that most of the glycolytic activity is present in the intestinal tissue of the adult parasites. This could suggest that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine. These findings provided useful information on the host-parasite interaction and the biology of this parasite, which can support the concerted efforts to develop better intervention strategies.
Na-FAR-1, a fatty acid- and retinol-binding protein, was expressed in bacteria, purified and crystallized. Crystals grew in two different morphologies under the same conditions.
Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning.
fatty acid- and retinol-binding proteins; parasitic nematodes; Necator americanus; Na-FAR-1
The collection of absorption and Raman spectroscopic data correlated with X-ray diffraction data allows investigators to understand the atomic structure as well as the electronic and vibrational characteristics of their samples, to identify transiently formed intermediates and to explore mechanistic questions. Raman spectroscopy instrumentation at beamline X26-C at the NSLS is currently available to the general user population.
Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of ‘mystery density’, i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data.
Raman; single-crystal spectroscopy; X-ray diffraction
LmACR2 from L. major is the first rhodanese-like enzyme directly involved in the reduction of arsenate and antimonate to be crystallized. Diffraction data have been collected to 1.99 Å resolution using synchrotron X-rays.
Arsenic is present in the biosphere owing either to the presence of pesticides and herbicides used in agricultural and industrial activities or to leaching from geological formations. The health effects of prolonged exposure to arsenic can be devastating and may lead to various forms of cancer. Antimony(V), which is chemically very similar to arsenic, is used instead in the treatment of leishmaniasis, an infection caused by the protozoan parasite Leishmania sp.; the reduction of pentavalent antimony contained in the drug Pentostam to the active trivalent form arises from the presence in the Leishmania genome of a gene, LmACR2, coding for the protein LmACR2 (14.5 kDa, 127 amino acids) that displays weak but significant sequence similarity to the catalytic domain of Cdc25 phosphatase and to rhodanese enzymes. For structural characterization, LmACR2 was overexpressed, purified to homogeneity and crystallized in a trigonal space group (P321 or P3121/P3221). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 Å and a = b = 111.0, c = 175.6 Å, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 Å.
arsenate/antimonate reductase; arsenic detoxification; rhodanese; Leishmania sp