The Molecular Mechanism of Shiga Toxin Stx2e Neutralization by a Single-domain Antibody Targeting the Cell Receptor-binding Domain

Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease.     

Use of audio biofeedback to reduce tibial impact accelerations during running

Visual biofeedback of tibial peak positive acceleration (PPA) during running has been used successfully as a method of gait retraining to reduce PPAs. Audio biofeedback generated from PPA may present a novel, portable alternative. The purpose of this study was to investigate the feasibility of using PPA-generated audio biofeedback to reduce PPAs while running. Nine runners were fitted with a wireless accelerometer on their left tibia. PPAs were recorded and a custom LabVIEW program was used to emit a single beep once the PPA reached a preset threshold. The numerical difference between this threshold and peak PPA during running was scaled to the pitch of the beep, such that a foot strike with greater PPA would result in a beep with higher pitch. Subjects were then instructed to (1) run without any beeps, and/or (2) keep the pitch of the beep as low as possible. Subjects participated in a single testing session that included a five minute warm-up and two rounds of biofeedback, which consisted of five minutes of running with biofeedback followed by five minutes of running without biofeedback. Subjects were able to significantly reduce PPAs during exposure to audio biofeedback. In addition, two rounds of biofeedback were sufficient for subjects to retain a reduction in PPAs without biofeedback. PPA-generated audio biofeedback therefore appears to be a feasible method of gait retraining to reduce PPAs in runners.     

Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans

Proteins secreted by Gram-positive bacteria are released into the culture medium with the obvious benefit that they usually retain their native conformation. This property makes these host cells potentially interesting for the production of recombinant proteins, as one can take full profit of established protocols for the purification of active proteins. Several state-of-the-art strategies to increase the yield of the secreted proteins will be discussed, using Streptomyces lividans as an example and compared with approaches used in some other host cells. It will be shown that approaches such as increasing expression and translation levels, choice of secretion pathway and modulation of proteins thereof, avoiding stress responses by changing expression levels of specific (stress) proteins, can be helpful to boost production yield. In addition, the potential of multi-omics approaches as a tool to understand the genetic background and metabolic fluxes in the host cell and to seek for new targets for strain and protein secretion improvement is discussed. It will be shown that S. lividans, along with other Gram-positive host cells, certainly plays a role as a production host for recombinant proteins in an economically viable way. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Paradoxical effects of rapamycin on experimental house dust mite-induced asthma

The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.     

S-layer nanoglycobiology of bacteria

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.     

Structural characterization and reactivity of gamma-octamolybdate functionalized by proline

The reaction of molybdate and dl-proline at pH 3.4 results in the formation of a Na₄[Mo₈O₂₆(proO)₂] · 22H₂O complex (pro = proline) in which two proline ligands are attached to molybdenum(VI) ions via monodentate coordination of the carboxylate groups. The structure of the complex was determined by single crystal X-ray diffraction and by combination of ¹H, ¹³C and ⁹⁵Mo NMR spectroscopy techniques in solution. The structure of the complex is strongly dependant on the pH. At native pH 3.4 the octamolybdate-type structure seems to be present in solution, but the increase of pH to 5.8 resulted in a rearrangement of the structure to a heptamolybdate-type structure. At physiological pH, the polyoxometalate framework was completely dissociated into the monomeric unit. The reactivity of the Na₄[Mo₈O₂₆(proO)₂] · 22H₂O towards the hydrolysis of ATP was tested at different pH values. While in solution at pH 3.4 the hydrolysis proceeded to yield AMP (adenosine monophosphate) and ADP (adenosine diphosphate) in nearly equal amounts, reaction mixture at pH 5.8 gave ADP as the only product of hydrolysis after 24 h of reaction. At neutral pH, the hydrolysis of ATP was slower, but it proceeded to yield 75% of ADP after 48 h of reaction.     

Indications towards a stereoselectivity of the salt-induced peptide formation reaction

Different reaction yields for l- and d-alanine in the salt-induced peptide formation (SIPF) reaction, differences in the circular dichroism spectra and the complex formation constants of the involved chlorocuprate complexes point at a stereoselective differentiation between the two stereoisomers in the SIPF reaction and give a possible explanation towards the origin of homochirality in the process of the origin of life. An explanation of the observed effects can for the time being only be based on assumptions but could possibly be related to the inherent chirality of the Cu^II ion as a central atom of the [CuCl(gly)(glyH₂)(H₂O)₂]⁺ complex due to parity violation in weak interactions and to amplification of chirality related to the structural properties of the complex.     

Easily polarizable proton transfer hydrogen bonds between the side chains of histidine and the carboxylic acid groups of glutamic and aspartic acid residues in proteins

The nature of the hydrogen bonds formed between glutamic acid and histidine residues between aspartic acid and histidine residues is studied by i.r. spectroscopy. These studies were performed with $\text{(}\text{l}\text{-Glu}\text{)}{}_{\mathrm{n}}\text{+(}\text{l}\text{-His}\text{)}{}_{\mathrm{n}}$ and with associates of monomeric Glu + His and Asp + His systems in solutions whereby these amino acids had protected α-amino and α-carboxylic groups. It is shown that the OH …N?^?O?H⁺N bonds are easily polarizable proton transfer hydrogen bonds. The residence time of the proton at the His is a little larger in the case of the Asp + His than in the case of the Glu + His systems. Polar environments shift this proton transfer equilibrium in favour of the proton limiting structure ^?O?H⁺N, and less polar ones in favour of the structure OH?N. These results demonstrate that the large proton polarizability of the hydrogen bonded system in the active centre of chymotrypsin is responsible for the charge shift caused by the substrate, and thus for the increase in reactivity of the serine residue and the catalytic activity of the enzyme.