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Author(s): Alexander E. S. Van Driessche (corresponding author) [1]; Nani Van Gerven [5, 6]; Paul H. H. Bomans [2, 3]; Rick R. M. Joosten [2, 3]; Heiner Friedrich [2, 3]; [...]

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes) (1-3). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia (1,4,5). Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF (6,7). Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 [Å.sup.3] pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism., Curli are bacterial surface appendages that have structural and physical characteristics of amyloid fibrils, best known from human degenerative diseases (7-9). However, the role of bacterial amyloids such as curli [...]

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria (1-3). The nanoscale repeat structure of the S-layer lattices and their self-assembly [...]

Surface exposure of antigens on bacterial cells can be critical for eliciting an effective antibody response. Therefore, we investigated the cellular localization of the fimbrial F17a-G receptor-binding domain, fused to the translocator domain of the AIDA-I autotransporter. Synthesis of the fusion protein, under the control of the L-arabinose-inducible [P.sub.BAD] promoter, was shown to permeabilize Escherichia coli K-12 and Salmonella enterica serovar Typhimurium cells. The presence of permeable cells interfered with several methods that are typically used to determine surface exposure of proteins, such as protease treatment and whole-cell ELISA. Double immunofluorescence microscopy, using a second antibody directed against [beta]-galactosidase, a bacterial protein expressed in the cytoplasm, allowed the simultaneous detection of antigen expression and permeability in individual cells.

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a ?-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca2+ ion coordination. A Ca2+-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology. [ABSTRACT FROM AUTHOR]

The article offers information on the effect of surface display of the fimbrial F17a-G receptor-binding domain through the autotransporter AIDA-I on the permeability of bacterial cells. It mentions that the surface exposure of bacterial cells affects the active antibody response of receptor-binding domains. It adds that the fusion of the fimbrial F17a-G and the translocator domain AIDA-I also brings great impact on the permeability of the bacterial cells, wherein the presence of permeable cells is interfered. Moreover, to be able to determine the antigen expression and permeability of individual cells, double immunofluorescence microscopy is utilized.

In vitro mimicking of the stimuli controlling in vivo-inducible bacterial promoters during infection of the host can be complex. Therefore, the use of the nematode Caenorhabditis elegans was evaluated, as a surrogate host to examine the expression of Salmonella enterica promoters. Green fluorescent protein (GFP+) was put under the control of the promoters of the pagC, mgtB, sseA, pgtE and fur genes of S. enterica. After infection of C. elegans with an S. enterica serovar Typhimurium vaccine strain expressing these constructs, clear bacterial expression of GFP+ was observed under the control of all five promoters, although significant expression was not always obtained in vitro. It is concluded that C. elegans constitutes a useful model system for the study of the in vivo expression of Salmonella promoters. [ABSTRACT FROM AUTHOR]