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In bacteria the formation of disulphide bonds is facilitated by a family of enzymes known as the disulphide bond forming (Dsb) proteins, which, despite low sequence homology, belong to the thioredoxin (TRX) superfamily. Among these enzymes is the disulphide bond-forming protein A (DsbA); a periplasmic thiol oxidase responsible for catalysing the oxidative folding of numerous cell envelope and secreted proteins. Pathogenic bacteria often contain diverse Dsb proteins with distinct functionalities commonly associated with pathogenesis. Here we investigate FtDsbA1, a DsbA homologue from the Gram-negative bacterium Francisella tularensis. Our study shows that FtDsbA1 shares a conserved TRX-like fold bridged by an alpha helical bundle showcased by all DsbA-like proteins. However, FtDsbA1 displays a highly unique variation on this structure, containing an extended and flexible N-terminus and secondary structural elements inserted within the core of the TRX fold itself, which together twist the overall DsbA-like architecture. Additionally, FtDsbA1 exhibits variations to the well conserved active site with an unusual dipeptide in the catalytic CXXC redox centre (CGKC), and a trans configuration for the conserved cis-proline loop, known for governing DsbA-substrate interactions. FtDsbA1’s redox properties are comparable to other DsbA enzymes, however, consistent with its atypical structure, functional analysis reveals FtDsbA1 has a high degree of substrate specificity suggesting a specialised role within F. tularensis’ oxidative folding pathway. Overall, this work underscores the remarkable malleability of the TRX catalytic core, a ubiquitous and ancestral protein fold. This not only contributes to broadening the structural and functional diversity seen within proteins utilising this core fold but will also enhance the accuracy of AI-driven protein structural prediction tools.

Summary: Disulfide bond (Dsb) oxidoreductases involved in oxidative protein folding govern bacterial survival and virulence. Over the past decade, oligomerization has emerged as a potential factor that dictates oxidoreductase activities. To investigate the role of oligomerization, we studied three Dsb-like ScsC oxidoreductases involved in copper resistance: the monomeric Salmonella enterica StScsC, and the trimeric Proteus mirabilis PmScsC and Caulobacter crescentus CcScsC. For copper sequestration, ScsC proteins must remain in the reduced form. However, all three ScsC proteins exhibit both dithiol oxidation and disulfide reduction activity, despite structural differences and previously reported limited in vitro activity. Most ScsC reductase activity relies on interactions with E. coli DsbD reductase, while oxidase activity depends on environmental oxidation. Interestingly, engineered monomeric PmScsC interacts effectively with the E. coli DsbB oxidase, at the partial expense of its reductase activity. These findings highlight oligomerization of oxidoreductases as a steric hindrance strategy to block undesirable upstream oxidative interactions.

Autotransporters are bacterial proteins that possess multiple activities associated with pathogenesis. Here the authors present the structure of the autotransporter subtilase Ssp from Serratia marcescens and show that its distinctive structural features are required for its cytotoxic function.

Abstract The formation of aggregates and biofilms enhances bacterial colonisation and infection progression by affording protection from antibiotics and host immune factors. Despite these advantages there is a trade-off, whereby bacterial dissemination is reduced. As such, biofilm development needs to be controlled to suit adaptation to different environments. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the assembly of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 variants from different Escherichia coli pathotypes. We show that specific interactions between amino acids on the contacting interfaces of adjacent Ag43 proteins drives a common mode of trans-association that leads to cell clumping. Furthermore, subtle variation of these interactions alters aggregation kinetics and the degree of compacting within cell clusters. Together, our structure–function investigation reveals an underlying molecular basis for variations in the density of bacterial communities.

Autotransporters are the core component of a molecular nano-machine that delivers cargo proteins across the outer membrane of Gram-negative bacteria. Part of the type V secretion system, this large family of proteins play a central role in controlling bacterial interactions with their environment by promoting adhesion to surfaces, biofilm formation, host colonization and invasion as well as cytotoxicity and immunomodulation. As such, autotransporters are key facilitators of fitness and pathogenesis and enable co-operation or competition with other bacteria. Recent years have witnessed a dramatic increase in the number of autotransporter sequences reported and a steady rise in functional studies, which further link these proteins to multiple virulence phenotypes. In this review we provide an overview of our current knowledge on classical autotransporter proteins, the archetype of this protein superfamily. We also carry out a phylogenetic analysis of their functional domains and present a new classification system for this exquisitely diverse group of bacterial proteins. The sixteen phylogenetic divisions identified establish sensible relationships between well characterized autotransporters and inform structural and functional predictions of uncharacterized proteins, which may guide future research aimed at addressing multiple unanswered aspects in this group of therapeutically important bacterial factors.

The formation of disulphide bonds is an essential step in the folding of many proteins that enter the secretory pathway; therefore, it is not surprising that eukaryotic and prokaryotic organisms have dedicated enzymatic systems to catalyse this process. In bacteria, one such enzyme is disulphide bond-forming protein A (DsbA), a thioredoxin-like thiol oxidase that catalyses the oxidative folding of proteins required for virulence and fitness. A large body of work on DsbA proteins, particularly Escherichia coli DsbA (EcDsbA), has demonstrated the key role that the Cys30-XX-Cys33 catalytic motif and its unique redox properties play in the thiol oxidase activity of this enzyme. Using mutational and functional analyses, here we identify that a set of charged residues, which form an acidic groove on the non-catalytic face of the enzyme, further modulate the activity of EcDsbA. Our high-resolution structures indicate that these residues form a water-mediated proton wire that can transfer protons from the bulk solvent to the active site. Our results support the view that proton shuffling may facilitate the stabilisation of the buried Cys33 thiolate formed during the redox reaction and promote the correct direction of the EcDsbA–substrate thiol–disulphide exchange. Comparison with other proteins of the same class and proteins of the thioredoxin-superfamily in general suggest that a proton relay system appears to be a conserved catalytic feature among this widespread superfamily of proteins. Furthermore, this study also indicates that the acidic groove of DsbA could be a promising allosteric site to develop novel DsbA inhibitors as antibacterial therapeutics.

Autotransporter proteins are localised to the bacterial surface and promote colonisation of host epithelial surfaces. Here, the authors present the crystal structure of autotransporter UpaB and show evidence for distinct binding sites for glycosaminoglycans and host fibronectin.

ABSTRACT Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection. IMPORTANCE Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infection (UTI), a disease of major significance to human health. Globally, the incidence of UPEC-mediated UTI is strongly associated with increasing antibiotic resistance, making this extremely common infection a major public health concern. In this report, we describe the regulatory, structural, functional, and immunogenic properties of a candidate UPEC vaccine antigen, IrmA. We demonstrate that IrmA is a small UPEC protein that forms a unique domain-swapped dimer with structural mimicry to several human cytokine receptors. We also show that IrmA binds to IL-2, IL-4, and IL-10, is strongly immunogenic in urosepsis patients, and is coexpressed with factors associated with biofilm formation. Overall, this work suggests a potential novel contribution for IrmA in UPEC infection.

Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.