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INTRODUCTION: While the European legislation states that laboratories of high-containment must be sealable for fumigation, they do not prescribe a minimal value for airtightness. Starting from a previous study in which we measured the airtightness in 4 BSL-3 laboratories with blower-door tests, we discuss the connection between airtightness and a successful decontamination by fumigation. METHODS: Biological indicators (BIs) consisting of spores of Geobacillus stearothermophilus on metal disks were laid out in laboratories of different levels of airtightness before performing a fumigation with aerosolized hydrogen peroxide using an automated device, according to the manufacturer’s instructions. RESULTS: Incubation of all BI disks placed in the facility with the highest level of airtightness showed complete inactivation of spores. However, in the facility with a lower level of airtightness, not all spores were inactivated. DISCUSSION: Air leaks might be a factor in the outcome of the decontamination of a room by fumigation, as seen in the laboratory with a lower level of airtightness, but other factors associated with the fumigation process might also be critical for a successful decontamination. CONCLUSION: We argue that a validation of the decontamination procedure, before first use or after important renovations of a laboratory of high-containment, is a more effective endpoint than reaching a predefined level of airtightness.

The human stomach pathogen Helicobacter pyloriattaches to healthy and inflamed gastric tissue through members of a paralogous family of ‘Helicobacter outer membrane proteins’ (Hops), including adhesins BabA, SabA, HopQ, LabA and HopZ. Hops share a conserved 25 kDa C-terminal region that is thought to form an autotransporter-like transmembrane domain. Instead, our results show that Hops contain a non-continuous transmembrane domain, composed of seven predicted β–strands at the C–terminus and one at the N-terminus. Folding and outer membrane localization of the C–terminal β–domain critically depends on a predicted transmembrane β-strand within the first 16 N-terminal residues. The N-terminus is shown to reside in the periplasm, and our crystal and small angle X-ray scattering structures for the SabA extracellular domain reveal a conserved coiled-coil stem domain that connects to transmembrane β-strand 1 and 2. Taken together, our data show that Hop adhesins represent a novel outer membrane protein topology encompassing an OmpA-like 8–stranded β-barrel that is interrupted by a 15–108 kDa domain inserted inside the first extracellular loop. The insertion of large, folded domains in an extracellular loop is unprecedented in bacterial outer membrane proteins and is expected to have important consequences on how these proteins reach the cell surface.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Crohn's disease (CD) is an inflammatory bowel disease characterized by an exaggerated immune response to commensal microbiota in the intestines of patients. Metagenomic studies have identified specific bacterial species and strains with increased prevalence in CD patients, amongst which is the adherent-invasiveEscherichia coli(AIEC) strain LF82. AIEC strains express long polar fimbriae (LPF), which are known to target Peyer's patches in a mouse CD model. Here, the recombinant production of a soluble, self-complemented construct of the LpfD protein ofE. coliLF82 is reported and it is demonstrated that it forms the adhesive tip subunit of LPF. The LpfD crystal reveals an N-terminal adhesin domain and a C-terminal pilin domain that connects the adhesin to the minor pilus subunit LpfE. Surface topology and sequence conservation in the adhesin domain hint at a putative receptor-binding pocket as found in theKlebsiella pneumoniaeMrkD andE. coliF17-G (GafD) adhesins. Immunohistostaining of murine intestinal tissue sections revealed that LpfD specifically binds to the intestinal mucosa and submucosa. LpfD binding was found to be resistant to treatment withO- orN-glycosidases, but was lost in collagenase-treated tissue sections, indicating the possible involvement of an intestinal matrix-associated protein as the LpfD receptor. LpfD strongly adhered to isolated fibronectin in anin vitroassay, and showed lower levels of binding to collagen V and laminin and no binding to collagens I, III and IV.

F4 (formerly K88) fimbriae from enterotoxigenic Escherichia coli are assembled via the FaeE/FaeD chaperone/usher pathway. The chaperone FaeE crystallizes in three crystal forms, all belonging to space group C2. Crystals of form 1 diffract to 2.3 A and have unit-cell parameters a = 195.7, b = 78.5, c = 184.6 A, beta = 102.2 degrees. X-ray data for crystal form 2 were collected to 2.7 A using an SeMet variant of FaeE. The crystals have unit-cell parameters a = 136.4, b = 75.7, c = 69.4 A, beta = 92.8 degrees. Crystals of form 3 were formed in a solution containing the FaeE-FaeG complex and diffract to 2.8 A. Unit-cell parameters are a = 109.7, b = 78.6, c = 87.8 A, beta = 96.4 degrees.