COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor – the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also L-fucose alone, bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called ‘secretors’

1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1H, 13C, 15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13C′, 13Cα, 13Cβ, 1HN and 15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens. This research was first published in Biomolecular NMR Assignments. © Springer Verlag

Specificity of Escherichia coli heat-Labile enterotoxin investigated by single-site mutagenesis and crystallography

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity

High-Resolution Crystal Structures Elucidate the Molecular Basis of Cholera Blood Group Dependence

Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1–1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera

Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

Cholera is a life-threatening disease in many countries, and new drugs are clearly needed. C-glycosidic antagonists may serve such a purpose. Here we report atomic-resolution crystal structures of three such compounds in complexes with the cholera toxin. The structures give unprecedented atomic details of the molecular interactions and show how the inhibitors efficiently block the GM1 binding site. These molecules are well suited for development into low-cost prophylactic drugs, due to their relatively easy synthesis and their resistance to glycolytic enzymes. One of the compounds links two toxin B-pentamers in the crystal structure, which may yield improved inhibition through the formation of toxin aggregates. These structures can spark the improved design of GM1 mimics, either alone or as multivalent inhibitors connecting multiple GM1-binding sites. Future developments may further include compounds that link the primary and secondary binding sites. Serving as decoys, receptor mimics may lessen symptoms while avoiding the use of antibiotics

Blood group antigen binding to the cholera toxin.

<p>(<b>A</b>) X-ray structure of cCTB in complex with A-penta-BGA (PDB ID: 5ELD); side and top views. The B-pentamer is colored by subunit, and the ligands are shown in stick representation. Blood group determinants are bound to the lateral side of the toxin (modeled into four of the five secondary binding sites), and galactose to the primary binding site (in three of the sites), facing the cell membrane. (<b>B-G</b>) Close-up views of the blood group antigen binding sites. Oligosaccharide residues are labeled in italics, amino acid residues in bold, and residues from neighboring subunits are indicated with a hash (#). (<b>B-C</b>) Interactions of the blood group A determinant with cCTB and ET CTB. Biotype-specific residues are highlighted in orange and green sticks. Water molecules are shown as red spheres, and hydrogen bonds are depicted as red dashed lines. (<b>D-E</b>) Electron density. <i>σ</i>_A-weighted <i>F</i>_o − <i>F</i>_c maps (green mesh; contoured at 3.0<i>σ</i>) are shown for the blood group A determinant and a human milk oligosaccharide. The maps were generated before insertion of the ligands. Circles indicate special features: the GlcNAc’s <i>N</i>-acetyl group, and the less-defined electron density for GalNAc in A-penta-BGA. (<b>F-G</b>) Interaction of the blood group H determinant with cCTB and ET CTB (two orientations). Yellow residues mark the reducing-end GlcNAc, α and β anomers are labeled. Water molecules are depicted as red spheres, hydrogen bonds represented by red dashed lines. In B, C, F and G, only those H-bonds are shown that have favorable angles, maximum bond lengths of 3.5 Å and that are conserved in all binding sites.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Structures and nomenclatures of the oligosaccharides in this article.

<p>Lewis-y blood group determinants H-tetra-BGA, B-penta-BGA, A-penta-BGA and the related human milk oligosaccharide A-penta-HMO. All of these have type-2 core structures. Note that H-tetra-HMO and A-penta-HMO were referred to as H-tetra and A-penta in our previous publications [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005567#ppat.1005567.ref027" target="_blank">27</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005567#ppat.1005567.ref029" target="_blank">29</a>]. Carbohydrate symbols follow the nomenclature of the Consortium for Functional Glycomics (Nomenclature Committee, Consortium for Functional Glycomics (functionalglycomics.org/static/consortium/Nomenclature.shtml); d-galactose (Gal)–yellow circle, <i>N</i>-acetylgalactosamine (GalNAc)–yellow square, d-glucose (Glc)–blue circle, <i>N</i>-acetylglucosamine (GlcNAc)–blue square, l-fucose (Fuc)–red triangle.</p