STRUCTURAL AND FUNCTIONAL CHARACTERISATION OF MUCUS ADHESION PROTEINS OF LACTOBACILLUS REUTERI

Mucus is the first point of contact between the gut microbiota and the host. Mucus adhesins are thought to be key mediators in the mucus adhesion of commensal Lactobacillus species. However, knowledge on the structural or functional basis of adhesin interaction with mucin glycoproteins, the main component of mucus, is limited. This work describes the biochemical and structural properties of two cell-surface proteins from Lactobacillus reuteri, the mucus-binding protein (MUB) and the Lar0958 protein, and their mucin binding ability. MUB from L. reuteri ATCC 53608 consists of 14 Mub repeats, six type 1 and eight type 2. Single and tandem Mub repeats were heterologously expressed and purified for structural and functional studies. The three-dimensional structure of the Mub type 1 MubRV was determined by X-ray crystallography and revealed two structural domains, B1 and B2. Furthermore, structural homology between MubRV and fibre-like adhesins of Gram-positive pathogens was identified. Small angle X-ray scattering experiments of single and tandem Mub repeats suggested an elongated structure of MUB in a ‘beads on a string’ arrangement. Functional studies of recombinant Mub repeats and the full-length native MUB isolated from Lactobacillus spent culture media, demonstrated binding to different mucins in vitro. Sugar inhibition experiments and glycan arrays suggested the involvement of sugar recognition in MUB protein binding to mucins. Lar0958 is a modular protein of six Lar0958 repeats present on the cell-surface of L. reuteri DSM 20016T. The crystal structure of a single recombinant Lar0958 repeat was solved at 1.5 Å, demonstrating a similar protein fold to Mub repeats. In addition, the Lar0958 repeat shows structural similarity to internalin proteins of the pathogen Listeria monocytogenes. Taken together these results provide new insights into the structural organisation of lactobacilli mucus adhesins and their interaction with mucins, highlighting similarities with Gram-positive adhesins of pathogenic bacteria

Suicide with One Cranial Gunshot by a .320 Caliber Pocket Revolver

Suicide using a firearm is the most commonly used method of committing suicide for men and women. In this paper, we present and discuss a suicide case in which an 86-year-old man shot himself using a .320 caliber pocket revolver. Proper crime scene investigation, recovery of the weapon used, and precise interpretation of autopsy findings play a fundamental role in determining the exact cause and manner of death. Accurate analysis of the injuries and a thorough knowledge of weapons and ballistics are essential for an adequate investigation in these unusual cases. To the best of our knowledge, this is the first report of a cranial gunshot inflicted by a .320 caliber pocket revolver

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer

Enzymatic and Chemoenzymatic Syntheses of Disialyl Glycans and Their Necrotizing Enterocolitis Preventing Effects

Necrotizing enterocolitis (NEC) is one of the most common and devastating intestinal disorders in preterm infants. Therapies to meet the clinical needs for this special and highly vulnerable population are extremely limited. A specific human milk oligosaccharide (HMO), disialyllacto-<i>N</i>-tetraose (DSLNT), was shown to contribute to the beneficial effects of breastfeeding as it prevented NEC in a neonatal rat model and was associated with lower NEC risk in a human clinical cohort study. Herein, gram-scale synthesis of two DSLNT analogs previously shown to have NEC preventing effect is described. In addition, four novel disialyl glycans have been designed and synthesized by enzymatic or chemoenzymatic methods. Noticeably, two disialyl tetraoses have been produced by enzymatic sialylation of chemically synthesized thioethyl β-disaccharides followed by removal of the thioethyl aglycon. Dose-dependent and single-dose comparison studies showed varying NEC-preventing effects of the disialyl glycans in neonatal rats. This study helps to refine the structure requirement of the NEC-preventing effect of disialyl glycans and provides important dose-dependent information for using DSLNT analogs as potential therapeutics for NEC prevention in preterm infants