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The SpyCatcher-SpyTag system has become a popular and versatile tool for protein ligation. It is based on a small globular protein (SpyCatcher) that binds to a 13-residue peptide (SpyTag), which subsequently leads to the formation of a covalent isopeptide bond. Thus, the reaction is essentially irreversible. Here, we describe how the SpyCatcher-SpyTag system can be used to label surface-exposed bacterial outer membrane proteins, e.g., for topology mapping or fluorescent time-course experiments. We cover using fluorescence measurements and microscopy to measure labeling efficiency using SpyCatcher fused with superfolder GFP in this chapter., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Transmembrane β-barrel proteins reside in the outer membrane of Gram-negative bacteria and are thus in direct contact with the environment. Because of that, they are involved in many key processes stretching from cellular survival to virulence. Hence, they are an attractive target for the development of novel antimicrobials, in addition to being of fundamental biological interest. To study this class of proteins, they are often required to be expressed in Escherichia coli. Recombinant expression of β-barrel proteins can be achieved using two fundamentally different strategies. The first alternative uses a complete coding sequence that includes a signal peptide for targeting the protein to its native cellular location, the bacterial outer membrane. The second alternative omits the signal peptide in the gene, leading to mislocalization and aggregation of the protein in the bacterial cytoplasm. These aggregates, called inclusion bodies, can be solubilized and the protein can be folded into its native form in vitro. In this chapter, we present example protocols for both strategies and discuss their advantages and disadvantages., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The outer membrane of Gram-negative bacteria is a specialized organelle conferring protection to the cell against various environmental stresses and resistance to many harmful compounds. The outer membrane has a number of unique features, including an asymmetric lipid bilayer, the presence of lipopolysaccharides and an individual proteome. The vast majority of the integral transmembrane proteins in the outer membrane belongs to the family of β-barrel proteins. These evolutionarily related proteins share a cylindrical, anti-parallel β-sheet core fold spanning the outer membrane. The loops and accessory domains attached to the β-barrel allow for a remarkable versatility in function for these proteins, ranging from diffusion pores and transporters to enzymes and adhesins. We summarize the current knowledge on β-barrel structure and folding and give an overview of their functions, evolution, and potential as drug targets., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this chapter, we review the computational approaches that have led to a new generation of vaccines in recent years. There are many alternative routes to develop vaccines based on the concept of reverse vaccinology. They all follow the same basic principles-mining available genome and proteome information for antigen candidates, and recombinantly expressing them for vaccine production. Some of the same principles have been used successfully for cancer therapy approaches. In this review, we focus on infectious diseases, describing the general workflow from bioinformatic predictions of antigens and epitopes down to examples where such predictions have been used successfully for vaccine development., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC, and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface., Importance: The secretion of trimeric autotransporters (TAA's) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rooke, Icke, Wells, Rossiter, Browning, Morris, Leo, Schütz, Autenrieth, Cunningham, Linke and Henderson.)

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat. The virulence factor intimin is essential for the attachment of pathogenic E. coli to the intestinal host cell. Intimin consists of four extracellular bacterial immunoglobulin-like (Big) domains, D00-D2, extending into the fifth lectin subdomain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of the elusive D00-D0 domains at 1.5 Å and D0-D1 at 1.8 Å resolution, which confirms that the passenger of intimin has five distinct domains. We describe that D00-D0 exhibits a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We conclude that D00 is a unique Big domain with a specific topology likely found in a broad range of other inverse autotransporters. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00-D0-D1, extending directly from the membrane.

Trimeric autotransporter adhesins (TAAs) comprise a group of virulence-related proteins in Gram-negative bacteria. Members of this family bind to extracellular matrix components such as collagen and fibronectin, but also they exhibit several other functions, such as conferring serum resistance and autoaggregation. Autoaggregation promoted by TAAs is homotypic and mediated by the sticky, globular head domains of these lollipop-like molecules. However, whether TAAs mediate heterotypic interactions (i.e., coaggregation) has not been studied. To address this question, we investigated the coaggregation of two model TAA groups: YadA from the enteropathogenic Yersiniae and the immunoglobulin-binding Eib proteins from Escherichia coli. To study TAA coaggregation, we coexpressed a fluorescent label together with a particular TAA and followed the aggregative interactions using fluorescence microscopy and quantified the interactions using a novel script implemented in Fiji. Our results show that there is coaggregation between some populations expressing different TAAs, which can be explained by relatively high sequence similarity between the interacting TAAs. Generally, the level of coaggregation correlated with the sequence similarity. However, some TAAs did not interact despite high sequence similarity, showing exclusion of bacteria producing a noncompatible TAA. These data demonstrate that TAAs can mediate bacterial coaggregation, but in some cases prevent coaggregation of bacteria with disparate TAAs. Our results have implications for the ecology of TAA-producing bacteria, where coaggregation may promote co-operation whereas exclusion might be an indication of competition., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface-exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin-like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli , Shigella spp., and the enteropathogenic Yersiniae . The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered "cytoskeletoxins" that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes., Competing Interests: The authors declare no conflict of interest.

Following publication of the original article [1], an error was reported in the tagging of Eugene H. Johnson and Remya R. Nair in the author group.

Background: Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI)., Results: ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein., Conclusions: Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.

Autotransporters, or type 5 secretion systems, are widespread surface proteins of Gram-negative bacteria often associated with virulence functions. Autotransporters consist of an outer membrane β-barrel domain and an exported passenger. In the poorly studied type 5d subclass, the passenger is a patatin-like lipase. The prototype of this secretion pathway is PlpD of Pseudomonas aeruginosa , an opportunistic human pathogen. The PlpD passenger is a homodimer with phospholipase A1 (PLA1) activity. Based on sequencing data, PlpD-like proteins are present in many bacterial species. We characterized the enzymatic activity, specific lipid binding and oligomeric status of PlpD homologs from Aeromonas hydrophila (a fish pathogen), Burkholderia pseudomallei (a human pathogen) and Ralstonia solanacearum (a plant pathogen) and compared these with PlpD. We demonstrate that recombinant type 5d-secreted patatin domains have lipase activity and form dimers or higher-order oligomers. However, dimerization is not necessary for lipase activity; in fact, by making monomeric variants of PlpD, we show that enzymatic activity slightly increases while protein stability decreases. The lipases from the intracellular pathogens A. hydrophila and B. pseudomallei display PLA2 activity in addition to PLA1 activity. Although the type 5d-secreted lipases from the animal pathogens bound to intracellular lipid targets, phosphatidylserine and phosphatidylinositol phosphates, hydrolysis of these lipids could only be observed for FplA of Fusobacterium nucleatum Yet, we noted a correlation between high lipase activity in type 5d autotransporters and intracellular lifestyle. We hypothesize that type 5d phospholipases are intracellularly active and function in modulation of host cell signaling events., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Yersinia ruckeri is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of Y. ruckeri but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to Y. ruckeri virulence. This review summarizes the knowledge on Y. ruckeri virulence factors. Understanding the molecular pathogenicity of Y. ruckeri will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.

Bacteria secrete proteins for different purposes such as communication, virulence functions, adhesion to surfaces, nutrient acquisition, or growth inhibition of competing bacteria. For secretion of proteins, Gram-negative bacteria have evolved different secretion systems, classified as secretion systems I through IX to date. While some of these systems consist of multiple proteins building a complex spanning the cell envelope, the type V secretion system, the subject of this review, is rather minimal. Proteins of the Type V secretion system are often called autotransporters (ATs). In the simplest case, a type V secretion system consists of only one polypeptide chain with a β-barrel translocator domain in the membrane, and an extracellular passenger or effector region. Depending on the exact domain architecture of the protein, type V secretion systems can be further separated into sub-groups termed type Va through e, and possibly another recently identified subtype termed Vf. While this classification works well when it comes to the architecture of the proteins, this is not the case for the function(s) of the secreted passenger. In this review, we will give an overview of the functions of the passengers of the different AT classes, shedding more light on the variety of functions carried out by type V secretion systems.

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process., (© 2019 John Wiley & Sons Ltd.)

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the β-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC , of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae , pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system ( tra ). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Transmembrane proteins in the outer membrane of Gram-negative bacteria are almost exclusively β-barrels. They are inserted into the outer membrane by a conserved and essential protein complex called the BAM (for β-barrel assembly machinery). In this commentary, we summarize current research into the mechanism of this protein complex and how it relates to type V secretion. Type V secretion systems are autotransporters that all contain a β-barrel transmembrane domain inserted by BAM. In type Vc systems, this domain is a homotrimer. We argue that none of the current models are sufficient to explain BAM function particularly regarding type Vc secretion. We also find that current models based on the well-studied model system Escherichia coli mostly ignore the pronounced differences in BAM composition between different bacterial species. We propose a more holistic view on how all OMPs, including autotransporters, are incorporated into the lipid bilayer., Competing Interests: Conflict of interest: All authors declare no conflicts of interest in this paper.

Many bacteria, both environmental and pathogenic, exhibit the property of autoaggregation. In autoaggregation (sometimes also called autoagglutination or flocculation), bacteria of the same type form multicellular clumps that eventually settle at the bottom of culture tubes. Autoaggregation is generally mediated by self-recognising surface structures, such as proteins and exopolysaccharides, which we term collectively as autoagglutinins. Although a widespread phenomenon, in most cases the function of autoaggregation is poorly understood, though there is evidence to show that aggregating bacteria are protected from environmental stresses or host responses. Autoaggregation is also often among the first steps in forming biofilms. Here, we review the current knowledge on autoaggregation, the role of autoaggregation in biofilm formation and pathogenesis, and molecular mechanisms leading to aggregation using specific examples., Competing Interests: Conflict of interest: All authors declare no conflicts of interest in this paper.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21 Gold (DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis., (© 2016 S. Karger AG, Basel.)

The Yersiniae are a group of Gram-negative coccobacilli inhabiting a wide range of habitats. The genus harbors three recognized human pathogens: Y. enterocolitica and Y. pseudotuberculosis, which both cause gastrointestinal disease, and Y. pestis, the causative agent of plague. These three organisms have served as models for a number of aspects of infection biology, including adhesion, immune evasion, evolution of pathogenic traits, and retracing the course of ancient pandemics. The virulence of the pathogenic Yersiniae is heavily dependent on a number of adhesin molecules. Some of these, such as the Yersinia adhesin A and invasin of the enteropathogenic species, and the pH 6 antigen of Y. pestis, have been extensively studied. However, genomic sequencing has uncovered a host of other adhesins present in these organisms, the functions of which are only starting to be investigated. Here, we review the current state of knowledge on the adhesin molecules present in the Yersiniae, and their functions and putative roles in the infection process., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane β-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the β-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: Breast reconstruction (BR) after mastectomy is widely recommended but there are significant variations in its application. The objective was to know the rate of BR in the Andalusian Public Health System (APHS), timing (immediate or delayed), surgical procedure, frequency of postoperative complications and their characteristics., Methods: We used the Minimum Basic Data Set of the APHS with personal data and identification of hospitals encrypted. We selected discharges for breast cancer and mastectomy in 2010-2013 and related readmissions of the same patients in 2010-2014. BR rates were calculated according to patient age and type of mastectomy. Timing of BR (immediate or delayed) and surgical techniques used were described. Postoperative complications were analyzed in the initial episode and in readmissions occurring within a minimum period of 2 years. BR failures were specifically studied., Results: We analyzed the information of 6,026 women, of which 4,412 met the inclusion criteria (basically, two years follow-up). The BR rate was 29% (22% immediate and 7% delayed) and reached 58% in women younger than 46 years. BR was performed by 27 of the 36 hospitals that practice mastectomies. Global percentage of postoperative complications was 18.6% for immediate BR, 12.1% for delayed BR and 7.9% for patients without BR. Failure occurred in 12.7% of immediate BR and 7.2% of delayed BR., Conclusions: In the Andalusian Public Health System the Breast reconstruction rate, is at a similar level to that reported nationally and in other countries.

Type V secretion denotes a variety of secretion systems that cross the outer membrane in Gram-negative bacteria but that depend on the Sec machinery for transport through the inner membrane. They are possibly the simplest bacterial secretion systems, because they consist only of a single polypeptide chain (or two chains in the case of two-partner secretion). Their seemingly autonomous transport through the outer membrane has led to the term "autotransporters" for various subclasses of type V secretion. In this chapter, we review the structure and function of these transporters and review recent findings on additional factors involved in the secretion process, which have put the term "autotransporter" to debate.

In this chapter, we review the computational approaches that have led to a new generation of vaccines in recent years. There are many alternative routes to develop vaccines based on the technology of reverse vaccinology. We focus here on bacterial infectious diseases, describing the general workflow from bioinformatic predictions of antigens and epitopes down to examples where such predictions have been used successfully for vaccine development.

The trimeric autotransporter adhesin Yersinia adhesin A is the prototype of the type Vc secretion systems. It is expressed by enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains, but not by Yersinia pestis. A characteristic trait of YadA is its modular composition and trimeric nature. YadA consists of an N-terminal passenger domain which is exposed on the bacterial cell surface. The translocation of this passenger onto the surface is facilitated by a C-terminal β-barrel domain which concomitantly anchors YadA into the outer membrane with three YadA monomers contributing to the formation of a single β-barrel. In Y. enterocolitica, but not Y. pseudotuberculosis, YadA is a decisive virulence factor and its deletion renders the bacteria virtually avirulent in mouse models of infection. This striking importance of YadA in infection may derive from its manifold functions in host cell interaction. Presumably the most important function of YadA is that it mediates adhesion to extracellular matrix components of eukaryotic host cells. Only tight adhesion allows for the injection of "anti-host" effector proteins via a type III secretion system into the host cell cytosol. These effector proteins enable Yersinia to subvert the host immune system in order to replicate and establish infection. YadA is also essential for the survival of Y. enterocolitica upon contact with serum, an important immune-evasion mechanism called serum resistance. To this end, YadA interacts with several components of the host complement system, the first line of immune defense. This review will summarize recent findings about the structure and biogenesis of YadA and its interactions with the host complement system., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Intimin and invasin are adhesins and central virulence factors of attaching and effacing bacteria, such as enterohaemorrhagic Escherichia coli, and enteropathogenic Yersiniae, respectively. These proteins are prototypes of a large family of adhesins distributed widely in Gram-negative bacteria. It is now evident that this protein family represents a previously unrecognized autotransporter secretion system, termed type Ve secretion. In contrast to classical autotransport, where the transmembrane β-barrel domain or translocation unit is C-terminal to the extracellular region or passenger domain, type Ve-secreted proteins have an inverted topology with the passenger domain C-terminal to the translocation unit; hence the term inverse autotransporter. This minireview covers the recent advances in elucidating the structure and biogenesis of inverse autotransporters., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a β-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract., (© 2014 Max Planck Society. Molecular Microbiology published by John Wiley & Sons Ltd.)

In addition to the cytoplasmic membrane, Gram-negative bacteria have a second lipid bilayer, the outer membrane, which is the de facto barrier between the cell and the extracellular milieu. Virtually all integral proteins of the outer membrane form β-barrels, which are inserted into the outer membrane by the BAM complex. Some outer membrane proteins, like the porins and trimeric autotransporter adhesins, are multimeric. In the former case, the porin trimer consists of three individual β-barrels, whereas in the latter, the single autotransporter β-barrel domain is formed by three separate polypeptides. This chapter reviews methods to investigate the folding and membrane insertion of multimeric OMPs and further explains the use of a BamA depletion strain to study the effects of the BAM complex on multimeric OMPs in E. coli.

The tendency of bacteria to assemble at oil-water interfaces can be utilized to create microbial recognition sites on the surface of polymer beads. In this work, two different groups of bacteria were first treated with acryloyl-functionalized chitosan and then used to stabilize an oil-in-water emulsion composed of cross-linking monomers that were dispersed in aqueous buffer. Polymerization of the oil phase followed by removal of the bacterial template resulted in well-defined polymer beads bearing bacterial imprints. Chemical passivation of chitosan and cell displacement assays indicate that the bacterial recognition on the polymer beads was dependent on the nature of the pre-polymer and the target bacteria. The functional materials for microbial recognition show great potential for constructing cell-cell communication networks, biosensors, and new platforms for testing antibiotic drugs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term 'autotransport' debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters.

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the β-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the β-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the β-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.

Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin Bartonella adhesin A (BadA) which is modularly constructed and consists of a head, a long and repetitive neck-stalk module with 22 repetitive neck/stalk repeats and a membrane anchor. The BadA head is crucial for bacterial adherence to host cells, binding to several extracellular matrix proteins and for the induction of vascular endothelial growth factor (VEGF) secretion. Here, we analysed the biological role of the BadA stalk in the infection process in greater detail. For this purpose, BadA head-bearing and headless deletion mutants with different lengths (containing one or four neck/stalk repeats in the neck-stalk module) were produced and functionally analysed for their ability to bind to fibronectin, collagen and endothelial cells and to induce VEGF secretion. Whereas a head-bearing short version (one neck/stalk element) of BadA lacks exclusively fibronectin binding, a substantially truncated headless BadA mutant was deficient for all of these biological functions. The expression of a longer headless BadA mutant (four neck/stalk repeats) restored fibronectin and collagen binding, adherence to host cells and the induction of VEGF secretion. Our data suggest that (i) the stalk of BadA is exclusively responsible for fibronectin binding and that (ii) both the head and stalk of BadA mediate adherence to collagen and host cells and the induction of VEGF secretion. This indicates overlapping functions of the BadA head and stalk., (© 2011 Blackwell Publishing Ltd.)

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.

The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bacteria of the Gram-negative genus Yersinia are environmentally ubiquitous. Three species are of medical importance: the intestinal pathogens Y. enterocolitica and Y. pseudotuberculosis, and the plague bacillus Y. pestis. The two former species, spread by contaminated food or water, cause a range of gastrointestinal symptoms and, rarely, sepsis. On occasion, the primary infection is followed by autoimmune sequelae such as reactive arthritis. Plague is a systemic disease with high mortality. It is a zoonosis spread by fleas, or more rarely by droplets from individuals suffering from pneumonic plague. Y. pestis is one of the most virulent of bacteria, and recent findings of antibiotic-resistant strains together with its potential use as a bioweapon have increased interest in the species. In addition to being significant pathogens in their own right, the yersiniae have been used as model systems for a number of aspects of pathogenicity. This chapter reviews the molecular mechanisms of adhesion in yersiniae. The enteropathogenic species share three adhesins: invasin, YadA and Ail. Invasin is the first adhesin required for enteric infection; it binds to β(1) integrins on microfold cells in the distal ileum, leading to the ingestion of the bacteria and allows them to cross the intestinal epithelium. YadA is the major adhesin in host tissues. It is a multifunctional protein, conferring adherence to cells and extracellular matrix components, serum and phagocytosis resistance, and the ability to autoagglutinate. Ail has a minor role in adhesion and serum resistance. Y. pestis lacks both invasin and YadA, but expresses several other adhesins. These include the pH 6 antigen and autotransporter adhesins. Also the plasminogen activator of Y. pestis can mediate adherence to host cells. Although the adhesins of the pathogenic yersiniae have been studied extensively, their exact roles in the biology of infection remain elusive.

Trimeric autotransporter adhesins (TAAs) are a family of secreted Gram-negative bacterial outer membrane (OM) proteins. These obligate homotrimeric proteins share a common molecular organisation, consisting of a N-terminal "passenger" domain followed by a C-terminal translocation unit/membrane anchor. All described TAAs act as adhesins. The passenger domain is responsible for specific adhesive and other activities of the protein and has a modular architecture. Its globular head domain(s), where ligands often bind, are projected away from the bacterial surface by an extended triple α-helical coiled coil stalk attached to the β-barrel anchor. The head domains appear to be constructed from a limited set of subdomains. The β-barrel anchor is the only part of the protein strictly conserved between family members. It appears that the extracellular export of the passenger does not require an external energy source or auxiliary proteins, though recent data indicate that an OM complex (the Bam complex) is involved in passenger domain secretion. The ability to bind to a variety of host molecules such as collagen, fibronectin, laminin or cell surface receptors via a structurally diverse elements suggests that TAAs have evolved a unique mechanism which closely links structure to folding and function.

The Yersinia adhesin YadA mediates the adhesion of the human enteropathogen Yersinia enterocolitica to collagens and other components of the extracellular matrix. Though YadA has been proposed to bind to a specific site in collagens, the exact binding determinants for YadA in native collagen have not previously been elucidated. We investigated the binding of YadA to collagen Toolkits, which are libraries of triple-helical peptides spanning the sequences of type II and III human collagens. YadA bound to many of them, in particular to peptides rich in hydroxyproline but with few charged residues. We were able to block the binding of YadA to collagen type IV with the triple-helical peptide (Pro-Hyp-Gly)(10), suggesting that the same site in YadA binds to triple-helical regions in network-forming collagens as well. We showed that a single Gly-Pro-Hyp triplet in a triple-helical peptide was sufficient to support YadA binding, but more than six triplets were required to form a tight YadA binding site. This is significantly longer than the case for eukaryotic collagen-binding proteins. YadA-expressing bacteria bound promiscuously to Toolkit peptides. Promiscuous binding could be advantageous for pathogenicity in Y. enterocolitica and, indeed, for other pathogenic bacteria. Many of the tightly binding peptides are also targets for eukaryotic collagen-binding proteins, and YadA was able to inhibit the interaction between selected Toolkit peptides and platelets. This leads to the intriguing possibility that YadA may interfere in vivo with host processes mediated by endogenous collagen-binding proteins.

We recently published a paper in Molecular Immunology (Leo and Goldman, 2009) showing that the Eib proteins from certain E. coli strains bind IgG Fc, in contrast to a previous report (Ghumra and Pleass, 2007). Richard J. Pleass has commented on our paper (Pleass, 2009) and expressed a series of unjustified misgivings about our conclusions. Here, we address the points raised by Pleass, and reassert our claim that the Eibs are indeed genuine receptors for IgG Fc., ((c) 2009 Elsevier Ltd. All rights reserved.)