The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Background The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. Results The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. Conclusions Cry6 proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities

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

Vertical heterogeneity of chromium as a proxy for reactive metallic contaminants in the pore water of a municipal solid waste landfill subject to leachate recirculation

As part of the experiment introduction sustainable landfill management (iDS), three pilot projects were instigated in which landfills are being stabilized through leachate recirculation and/or aeration. The ultimate goal of these projects is to improve the leachate quality until it complies with previously derived environmental protection criteria. The hydrology and leachate transport of one of the three landfills – a municipal solid waste landfill being subjected to leachate recirculation - has been difficult to characterize. To further elucidate transport processes in this landfill, two vadose zone monitoring systems (VMS) have been installed. The VMS are installed at a 45 degree incline, and allow for pore water sampling at 0.4 metre depth intervals between 3 – 6.5 metres and 9.6 – 13 metres below the landfill surface. These systems provide a unique opportunity to collect and analyse in-situ pore water samples for a multitude of parameters of importance for contaminant speciation and transport, thereby providing valuable information on the vertical heterogeneity within the landfill. Starting from July 2022, the VMS are sampled every 3 months and analysed for parameters relevant for understanding contaminant availability and transport throughout the landfill, i.e. contaminant concentrations, concentrations of reactive colloidal particles, pH, and electrical conductivity. A separate paper focuses on the amount and composition of dissolved organic matter in the pore water as a function of depth in the landfill. This paper builds further on that study and describes how these parameters ultimately govern the speciation and vertical distribution of contaminants and its change with time, using Chromium as a proxy for contaminant behaviour

Additional file 3: Figure S3. of The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Modeled L259D mutant. The full-length structure (5KUC, magenta) is overlaid with the L259D model (cyan). The inset illustrates the putative transmembrane loop region with residue 259 shown in stick display. Loops are colored as above for clarity while, for other regions, the full-length structure is dark gray and the modeled mutant is light gray. (PPTX 737 kb

Additional file 2: Figure S2. of The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Ab initio models of Cry6Aa. The five models constructed with reference to database information (db) and five models constructed entirely de novo (dn) are shown. The predicted N-terminal domains are shown in light gray and marked “N,” while the C-terminal domains are shown in black and marked “C.” (PPTX 774 kb

Additional file 4: Figure S4. of The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Western blot of wild-type and L259D mutant Cry6Aa. Expression of the wild-type Cry6Aa (WT) and the L259D mutant confirmed by western blotting. (PPTX 1138 kb