Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1-β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

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The Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin of enteric yersiniae. It consists of three major domains: a head mediating adherence to host cells, a stalk involved in serum resistance, and an anchor that forms a membrane pore and is responsible for the autotransport function. The anchor contains a glycine residue, nearly invariant throughout trimeric autotransporter adhesins, that faces the pore lumen. To address the role of this glycine, we replaced it with polar amino acids of increasing side chain size and expressed wild-type and mutant YadA in Escherichia coli. The mutations did not impair the YadA-mediated adhesion to collagen and to host cells or the host cell cytokine production, but they decreased the expression levels and stability of YadA trimers with increasing side chain size. Likewise, autoagglutination and resistance to serum were decreased in these mutants. We found that the periplasmic protease DegP is involved in the degradation of YadA and that in an E. coli degP deletion strain, mutant versions of YadA were expressed almost to wild-type levels. We conclude that the conserved glycine residue affects both the export and the stability of YadA and consequently some of its putative functions in pathogenesis.

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80+, 11% of CD11c+, 7% of CD49b+, 5% of Gr1+ cells, 2.3% of CD19+, and 2.6% of CD3+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19+CD21+CD23+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

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The capacity of pathogenic microorganisms to adhere to host cells and avoid clearance by the host immune system is the initial and most decisive step leading to infections. Bacteria have developed different strategies to attach to diverse host surface structures. One important strategy is the adhesion to extracellular matrix (ECM) proteins (e.g., collagen, fibronectin, laminin) that are highly abundant in connective tissue and basement membranes. Gram-negative bacteria express variable outer membrane proteins (adhesins) to attach to the host and to initiate the process of infection. Understanding the underlying molecular mechanisms of bacterial adhesion is a prerequisite for targeting this interaction by "anti-ligands" to prevent colonization or infection of the host. Future development of such "anti-ligands" (specifically interfering with bacteria-host matrix interactions) might result in the development of a new class of anti-infective drugs for the therapy of infections caused by multidrug-resistant Gram-negative bacteria. This review summarizes our current knowledge about the manifold interactions of adhesins expressed by Gram-negative bacteria with ECM proteins and the use of this information for the generation of novel therapeutic antivirulence strategies.

The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed-and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (؊2.66 hits per million sample reads/defined daily dose; P ‫؍‬ 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens.

Invasin and intimin are major virulence factors of enteropathogenic Yersiniae and Escherichia coli, mediating invasion into and intimate adherence to host cells, respectively. Several studies have hinted that extracellular portion of these homologous proteins might be exported via an autotransport mechanism, but rigorous experimental proof has been lacking. Here, we present a topology model for invasin and intimin, consistent with the hypothesis that the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. We confirmed this topology model by inserting epitope tags into the loops of the β-barrel. We further show that obstructing the pore of β-barrel hinders the export of the passenger domain. As for classical autotransport, the biogenesis of invasin and intimin is dependent on the Bam complex and the periplasmic chaperone SurA, whereas the chaperone/protease DegP is involved in quality control. However, compared to classical autotransporters (Type Va secretion), the domain structure of intimin and invasin is inverted. We conclude that proteins of the intimin and invasin family constitute a novel group of autotransported proteins, and propose that this class of autotransporters be termed Type Ve secretion.