Psychoactive and other ceremonial plants from a 2,000-year-old Maya ritual deposit at Yaxnohcah, Mexico.

For millennia, healing and psychoactive plants have been part of the medicinal and ceremonial fabric of elaborate rituals and everyday religious practices throughout Mesoamerica. Despite the essential nature of these ritual practices to the societal framework of past cultures, a clear understanding of the ceremonial life of the ancient Maya remains stubbornly elusive. Here we record the discovery of a special ritual deposit, likely wrapped in a bundle, located beneath the end field of a Late Preclassic ballcourt in the Helena complex of the Maya city of Yaxnohcah. This discovery was made possible by the application of environmental DNA technology. Plants identified through this analytical process included Ipomoea corymbosa (xtabentun in Mayan), Capsicum sp. (chili pepper or ic in Mayan), Hampea trilobata (jool), and Oxandra lanceolata (chilcahuite). All four plants have recognized medicinal properties. Two of the plants, jool and chilcahuite, are involved in artifact manufacture that have ceremonial connections while chili peppers and xtabentun have been associated with divination rituals. Xtabentun (known to the Aztecs as ololiuhqui) produces highly efficacious hallucinogenic compounds and is reported here from Maya archaeological contexts for the first time.

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

Environmental DNA reveals arboreal cityscapes at the Ancient Maya Center of Tikal.

Tikal, a major city of the ancient Maya world, has been the focus of archaeological research for over a century, yet the interactions between the Maya and the surrounding Neotropical forests remain largely enigmatic. This study aimed to help fill that void by using a powerful new technology, environmental DNA analysis, that enabled us to characterize the site core vegetation growing in association with the artificial reservoirs that provided the city water supply. Because the area has no permanent water sources, such as lakes or rivers, these reservoirs were key to the survival of the city, especially during the population expansion of the Classic period (250-850 CE). In the absence of specific evidence, the nature of the vegetation surrounding the reservoirs has been the subject of scientific hypotheses and artistic renderings for decades. To address these hypotheses we captured homologous sequences of vascular plant DNA extracted from reservoir sediments by using a targeted enrichment approach involving 120-bp genetic probes. Our samples encompassed the time before, during and after the occupation of Tikal (1000 BCE-900 CE). Results indicate that the banks of the ancient reservoirs were primarily fringed with native tropical forest vegetation rather than domesticated species during the Maya occupation.

Molecular genetic and geochemical assays reveal severe contamination of drinking water reservoirs at the ancient Maya city of Tikal.

Understanding civilizations of the past and how they emerge and eventually falter is a primary research focus of archaeological investigations because these provocative data sets offer critical insights into long-term human behavior patterns, especially in regard to land use practices and sustainable environmental interactions. The ancient Maya serve as an intriguing example of this research focus, yet the details of their spectacular emergence in a tropical forest environment followed by their eventual demise have remained enigmatic. Tikal, one of the foremost of the ancient Maya cities, plays a central role in this discussion because of its sharp population decline followed by abandonment during the late 9th century CE. Our results, based on geochemical and molecular genetic assays on sediments from four of the main reservoirs, reveal that two of the largest reservoirs at Tikal, essential for the survival of the city during the dry seasons, were contaminated with high levels of mercury, phosphate and cyanobacteria known to produce deadly toxins. Our observations demonstrate severe pollution problems at a time when episodes of climatic aridity were prevalent. This combination of catastrophic events clearly threatened the sustainability of the city and likely contributed to its abandonment.

Tissue Responses to Shiga Toxin in Human Intestinal Organoids.

BACKGROUND & AIMS: Shiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell-derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. METHODS: In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice. RESULTS: Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal-epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. CONCLUSIONS: Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.

Electroosmotic flow driven microfluidic device for bacteria isolation using magnetic microbeads.

The presence of bacterial pathogens in water can lead to severe complications such as infection and food poisoning. This research proposes a point-of-care electroosmotic flow driven microfluidic device for rapid isolation and detection of E. coli in buffered solution (phosphate buffered saline solution). Fluorescent E. coli bound to magnetic microbeads were driven through the microfluidic device using both constant forward flow and periodic flow switching at concentrations ranging from 2 × 105 to 4 × 107 bacteria/mL. A calibration curve of fluorescent intensity as a function of bacteria concentration was created using both constant and switching flow, showing an increase in captured fluorescent pixel count as concentration increases. In addition, the use of the flow switching resulted in a significant increase in the capture efficiency of E. coli, with capture efficiencies up to 83% ± 8% as compared to the constant flow capture efficiencies (up to 39% ± 11%), with a sample size of 3 µL. These results demonstrate the improved performance associated with the use of the electroosmotic flow switching system in a point-of-care bacterial detection assay.

Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli.

Infection with Shiga toxin (Stx) producing Escherichia coli O157:H7 can cause the potentially fatal complication hemolytic uremic syndrome, and currently only supportive therapy is available. Lack of suitable animal models has hindered study of this disease. Induced human intestinal organoids (iHIOs), generated by in vitro differentiation of pluripotent stem cells, represent differentiated human intestinal tissue. We show that iHIOs with addition of human neutrophils can model E. coli intestinal infection and innate cellular responses. Commensal and O157:H7 introduced into the iHIO lumen replicated rapidly achieving high numbers. Commensal E. coli did not cause damage, and were completely contained within the lumen, suggesting defenses, such as mucus production, can constrain non-pathogenic strains. Some O157:H7 initially co-localized with cellular actin. Loss of actin and epithelial integrity was observed after 4 hours. O157:H7 grew as filaments, consistent with activation of the bacterial SOS stress response. SOS is induced by reactive oxygen species (ROS), and O157:H7 infection increased ROS production. Transcriptional profiling (RNAseq) demonstrated that both commensal and O157:H7 upregulated genes associated with gastrointestinal maturation, while infection with O157:H7 upregulated inflammatory responses, including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment, and infection with O157:H7 resulted in recruitment of human neutrophils into the iHIO tissue.

Plasmonics-Based Detection of Virus Using Sialic Acid Functionalized Gold Nanoparticles.

Biosensor for the detection of virus was developed by utilizing plasmonic peak shift phenomenon of the gold nanoparticles and viral infection mechanism of hemagglutinin on virus and sialic acid on animal cells. The plasmonic peak of the colloidal gold nanoparticles changes with the aggregation of the particles due to the plasmonic interaction between nearby particles and the color of the colloidal nanoparticle solution changes from wine red to purple. Sialic acid reduced and stabilized colloidal gold nanoparticle aggregation is induced by the addition of viral particles in the solution due to the hemagglutinin-sialic acid interaction. In this work, sialic acid reduced and stabilized gold nanoparticles (d = 20.1 ± 1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B virus species. The detection limit of the virus dilution (hemagglutinination assay titer, 512) was shown to be 0.156 vol% and the upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.

Development of a point-of-care diagnostic for influenza detection with antiviral treatment effectiveness indication.

Currently, diagnosis of influenza is performed either through tedious polymerase chain reaction (PCR) or through rapid antigen detection assays. The rapid antigen detection assays available today are highly specific but not very sensitive, and most importantly, lack the ability to show if the strain of influenza detected is susceptible to antiviral agents, such as Tamiflu and Relenza. The ability to rapidly determine if a patient has an infectious disease and what type of treatment the infection will respond to, would significantly reduce the treatment decision time, shorten the impact of symptoms, and minimize transfer to others. In this study, a novel, point-of-care style µPAD (microfluidic paper-based diagnostic) for influenza has been developed with the ability to determine antiviral susceptibility of the strain for treatment decision. The assay exploits the enzymatic activity of surface proteins present on all influenza strains, and potential false positive responses can be mitigated. A sample can be added to the device, distributed to 4 different reagent zones, and development of the enzymatic substrate under different buffer conditions takes place on bottom of the device. Analysis can be performed by eye or through a colorimetric image analysis smartphone application.

Shiga Toxin Mediated Neurologic Changes in Murine Model of Disease.

Seizures and neurologic involvement have been reported in patients infected with Shiga toxin (Stx) producing E. coli, and hemolytic uremic syndrome (HUS) with neurologic involvement is associated with more severe outcome. We investigated the extent of renal and neurologic damage in mice following injection of the highly potent form of Stx, Stx2a, and less potent Stx1. As observed in previous studies, Stx2a brought about moderate to acute tubular necrosis of proximal and distal tubules in the kidneys. Brain sections stained with hematoxylin and eosin (H&E) appeared normal, although some red blood cell congestion was observed. Microglial cell responses to neural injury include up-regulation of surface-marker expression (e.g., Iba1) and stereotypical morphological changes. Mice injected with Stx2a showed increased Iba1 staining, mild morphological changes associated with microglial activation (thickening of processes), and increased microglial staining per unit area. Microglial changes were observed in the cortex, hippocampus, and amygdala regions, but not the nucleus. Magnetic resonance imaging (MRI) of Stx2a-treated mice revealed no hyper-intensities in the brain, although magnetic resonance spectroscopy (MRS) revealed significantly decreased levels of phosphocreatine in the thalamus. Less dramatic changes were observed following Stx1 challenge. Neither immortalized microvascular endothelial cells from the cerebral cortex of mice (bEnd.3) nor primary human brain microvascular endothelial cells were found to be susceptible to Stx1 or Stx2a. The lack of susceptibility to Stx for both cell types correlated with an absence of receptor expression. These studies indicate Stx causes subtle, but identifiable changes in the mouse brain.

AB5 Preassembly Is Not Required for Shiga Toxin Activity.

UNLABELLED: Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5 toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5 toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5 complex is not required for cellular toxicity or in vivo toxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenic Escherichia coli is considered to be an AB5 heterohexamer; however, no known mechanisms ensure AB5 assembly. Stx released by E. coli is not in the AB5 conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5 assembly is a potential treatment for Stx-associated illnesses. IMPORTANCE: Complications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.

Single Amino Acid Polymorphisms of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function.

Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one µg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.

Glycolipid binding preferences of Shiga toxin variants.

The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx), an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA) to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

Pertussis toxin B-pentamer mediates intercellular transfer of membrane proteins and lipids.

Pertussis toxin (PTx) is the major virulence factor of Bordetella pertussis. The enzymatic or active (A) subunit inactivates host G protein coupled receptor (GPCR) signaling pathways. The non-enzymatic binding (B) subunit also mediates biological effects due to lectin-like binding characteristics, including the induction of T cell receptor (TCR) signaling and subsequent down-regulation of chemokine receptor expression. Here we report another activity attributable to PTxB, facilitating transfer of membrane material between mammalian cells. This activity does not require the TCR, and does not require cell-to-cell contact or cellular aggregation. Rather, membrane vesicles are transferred from donor to recipient cells in a toxin-dependent fashion. Membrane transfer occurs in different cell types, including cultured human T cells, CHO cells, and human primary peripheral blood mononuclear cells. Transfer involves both lipid and integral membrane proteins, as evidenced by the transfer of T and B cell-specific receptor molecules to other PBMCs. Interestingly, membrane transfer activity is a property that PTx shares with some, but not all, cell-aggregating lectins that are mitogenic for human T cells, and appears to be related to the ability to bind certain host cell glycolipids. This phenomenon may represent another mechanism by which pertussis toxin disrupts mammalian intra- and inter-cellular signaling.

Failure of manganese to protect from Shiga toxin.

Shiga toxin (Stx), the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S) both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.

Binding of Pk-trisaccharide analogs of globotriaosylceramide to Shiga toxin variants.

The two major forms of Shiga toxin, Stx1 and Stx2, use the glycolipid globotriaosylceramide (Gb3) as their cellular receptor. Stx1 primarily recognizes the Pk-trisaccharide portion and has three Pk binding sites per B monomer. The Stx2a subtype requires glycolipid residues in addition to Pk. We synthesized analogs of Pk to examine the binding preferences of Stx1 and Stx2 subtypes a to d. Furthermore, to determine how many binding sites must be engaged, the Pk analogues were conjugated to biotinylated mono- and biantennary platforms, allowing for the display of two to four Pk analogues per streptavidin molecule. Stx binding to Pk analogues immobilized on streptavidin-coated plates was assessed by enzyme-linked immunosorbent assay (ELISA). Stx1, but not the Stx2 subtypes, bound to native Pk. Stx2a and Stx2c bound to the Pk analog with a terminal GalNAc (NAc-Pk), while Stx1, Stx2b, and Stx2d did not bind to this analog. Interestingly, the purified Stx2d B subunit bound to NAc-Pk, suggesting that the A subunit of Stx2d interferes with binding. Disaccharide analogs (Galα1-4Gal, GalNAcα1-4Gal, and Galα1-4GalNAc) did not support the binding of any of the Stx forms, indicating that the trisaccharide is necessary for binding. Studies with monoantennary and biantennary analogs and mixtures suggest that Stx1, Stx2a, and Stx2c need to engage at least three Pk analogues for effective binding. To our knowledge, this is the first study examining the minimum number of Pk analogs required for effective binding and the first report documenting the role of the A subunit in influencing Stx2 binding.

Capture of uropathogenic E. coli by using synthetic glycan ligands specific for the pap-pilus.

Biotinylated mono- and biantennary di-/trisaccharides were synthesized to evaluate their ability to capture E. coli strains that express pilus types with different receptor specificities. The synthesized biotinylated di-/trisaccharides contain Galα(1→4)Gal, Galα(1→4)GalNHAc, GalNHAcα(1→4)Gal, Galα(1→4)Galß(1→4)Glc and GalNHAcα(1→4)Galß(1→4)Glc as carbohydrate epitopes. These biotinylated oligosaccharides were immobilized on streptavidin-coated magnetic beads, and incubated with different strains of live E. coli. Capturing ability was assessed by using a luciferase assay that detects bacterial ATP. The trisaccharides containing Galα(1→4)Galß(1→4)Glc and the disaccharides containing Galα(1→4)Gal as the epitopes exhibited strong capturing ability for uropathogenic E. coli strains with the pap pilus genotype, including CFT073, J96 and J96 pilE. The same ligands failed to capture E. coli strains with fim, prs, or foc genotypes. Uropathogenic CFT073 was also captured moderately by biantennary disaccharides containing a GalNHAc moiety at the reducing end; however, other saccharides containing GalNHAc at the nonreducing end did not capture the CFT073 strain. These synthetic glycoconjugates could potentially be adapted as rapid diagnostic agents to differentiate between different E. coli pathovars.

Colorimetric viral detection based on sialic acid stabilized gold nanoparticles.

Sialic acid reduced and stabilized gold nanoparticles (d=20.1±1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid for colorimetric detection of influenza virus. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B/Victoria and influenza B/Yamagata. Virus dilution (hemagglutination assay titer, 512) of 0.156 vol% was readily detected. The upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.

Mechanistic insight into pertussis toxin and lectin signaling using T cells engineered to express a CD8α/CD3ζ chimeric receptor.

Mammalian cell-surface receptors typically display N- or O-linked glycans added post-translationally. Plant lectins such as phytohemagluttinin (PHA) can activate the T cell receptor (TCR) and other cell-surface receptors by binding to glycans and initiating receptor cross-linking. Pathogenic microorganisms such as Bordetella pertussis also express proteins with lectin-like activities. Similar to plant lectins, pertussis toxin (PTx) can activate the TCR and bind to a variety of glycans. However, whether the lectin-like activity of PTx is responsible for its ability to activate TCR signaling has not been formally proven. Here we examined the ability of PTx and a panel of lectins to activate the TCR or a CD8α/CD3ζ chimeric receptor (termed CD8ζ). We demonstrate that CD8ζ rescues PTx-induced signaling events lacking in TCR null cells. This result indicates that CD8ζ can substitute for TCR and supports the hypothesis that PTxB (functioning as a lectin) stimulates signaling via receptor cross-linking rather than by binding to a specific epitope on the TCR. Moreover, PTx is able to activate signaling by binding either N-linked or O-linked glycan-modified receptors as the TCR displays N-linked glycans while CD8ζ displays O-linked glycans. Finally, studies with a diverse panel of lectins indicate that the signaling activity of the lectins does not always correlate with the biochemical reports of ligand preferences. Comparison of lectin signaling through TCR or CD8ζ allows us to better define the structural and functional properties of lectin-glycan interactions using a biologically based signaling readout.

Shiga toxin binding to glycolipids and glycans.

BACKGROUND: Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable. METHODOLOGY: We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells. RESULTS: By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells. CONCLUSIONS: Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED(50)) of protein synthesis was about 10(-11) M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.