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Aims: DsbA catalyzes disulfide bond formation in secreted and outer membrane proteins in bacteria. In pathogens, DsbA is a major facilitator of virulence constituting a target for antivirulence antimicrobial development. However, many pathogens encode multiple and diverse DsbA enzymes for virulence factor folding during infection. The aim of this study was to determine whether our recently identified inhibitors of Escherichia coli K-12 DsbA can inhibit the diverse DsbA enzymes found in two important human pathogens and attenuate their virulence. Results: DsbA inhibitors from two chemical classes (phenylthiophene and phenoxyphenyl derivatives) inhibited the virulence of uropathogenic E. coli and Salmonella enterica serovar Typhimurium, encoding two and three diverse DsbA homologues, respectively. Inhibitors blocked the virulence of dsbA null mutants complemented with structurally diverse DsbL and SrgA, suggesting that they were not selective for prototypical DsbA. Structural characterization of DsbA-inhibitor complexes showed that compounds from each class bind in a similar region of the hydrophobic groove adjacent to the Cys30-Pro31-His32-Cys33 (CPHC) active site. Modeling of DsbL- and SrgA-inhibitor interactions showed that these accessory enzymes could accommodate the inhibitors in their different hydrophobic grooves, supporting our in vivo findings. Further, we identified highly conserved residues surrounding the active site for 20 diverse bacterial DsbA enzymes, which could be exploited in developing inhibitors with a broad spectrum of activity. Innovation and Conclusion: We have developed tools to analyze the specificity of DsbA inhibitors in bacterial pathogens encoding multiple DsbA enzymes. This work demonstrates that DsbA inhibitors can be developed to target diverse homologues found in bacteria. Antioxid. Redox Signal. 29, 653–666.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

Environmental polarity is an important factor that drives biomolecular interactions to regulate cell function. Herein, a general method of using the fluorogenic probe NTPAN-MI is reported to quantify the subcellular polarity change in response to protein unfolding. NTPAN-MI fluorescence is selectively activated upon labeling unfolded proteins with exposed thiols, thereby reporting on the extent of proteostasis. NTPAN-MI also reveals the collapse of the host proteome caused by influenza A virus infection. The emission profile of NTPAN-MI contains information of the local polarity of the unfolded proteome, which can be resolved through spectral phasor analysis. Under stress conditions that disrupt different checkpoints of protein quality control, distinct patterns of dielectric constant distribution in the cytoplasm can be observed. However, in the nucleus, the unfolded proteome was found to experience a more hydrophilic environment across all the stress conditions, indicating the central role of nucleus in the stress response process.

The human pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) contains a complex disulfide bond (Dsb) catalytic machinery. This machinery encompasses multiple Dsb thiol-disulfide oxidoreductases that mediate oxidative protein folding and a less-characterized suppressor of copper sensitivity (scs) gene cluster, associated with increased tolerance to copper. To better understand the function of the Salmonella Scs system, here we characterized two of its key components, the membrane protein ScsB and the periplasmic protein ScsC. Our results revealed that these two proteins form a redox pair in which the electron transfer from the periplasmic domain of ScsB (n-ScsB) to ScsC is thermodynamically driven. We also demonstrate that the Scs reducing pathway remains separate from the Dsb oxidizing pathways and thereby avoids futile redox cycles. Additionally, we provide new insight into the molecular mechanism underlying Scs-mediated copper tolerance in Salmonella. We show that both ScsB and ScsC can bind toxic copper(I) with femtomolar affinities and transfer it to the periplasmic copper metallochaperone CueP. Our results indicate that the Salmonella Scs machinery has evolved a dual mode of action, capable of transferring reducing power to the oxidizing periplasm and protecting against copper stress by cooperating with the cue regulon, a major copper resistance mechanism in Salmonella. Overall, these findings expand our understanding of the functional diversity of Dsb-like systems, ranging from those mediating oxidative folding of proteins required for infection to those contributing to defense mechanisms against oxidative stress and copper toxicity, critical traits for niche adaptation and survival.

Graphical abstract, The disulfide bond (DSB) forming system and in particular DsbA, is a key bacterial oxidative folding catalyst. Due to its role in promoting the correct assembly of a wide range of virulence factors required at different stages of the infection process, DsbA is a master virulence rheostat, making it an attractive target for the development of new virulence blockers. Although DSB systems have been extensively studied across different bacterial species, to date, little is known about how DsbA oxidoreductases are able to recognize and interact with such a wide range of substrates. This review summarizes the current knowledge on the DsbA enzymes, with special attention on their interaction with the partner oxidase DsbB and substrates associated with bacterial virulence. The structurally and functionally diverse set of bacterial proteins that rely on DsbA-mediated disulfide bond formation are summarized. Local sequence and secondary structure elements of these substrates are analyzed to identify common elements recognized by DsbA enzymes. This not only provides information on protein folding systems in bacteria but also offers tools for identifying new DsbA substrates and informs current efforts aimed at developing DsbA targeted anti-microbials.

Bacterial aggregates and biofilms allow bacteria to colonise a diverse array of surfaces that can ultimately lead to infections, where the protection they afford permits bacteria to resist anti-microbials and host immune factors. Despite these advantages there is a trade-off, whereby bacterial spread is reduced. As such, biofilm development needs to be regulated appropriately to suit the required niche. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the formation of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 homologues from diverse pathogenic Escherichia strains. We reveal a common mode of trans-association that leads to cell clumping and show that subtle variations in these interactions governs their aggregation kinetics. Our in depth investigation reveals an underlying molecular basis for the ‘tuning’ of bacterial aggregation.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Enterococcus faecalisis an opportunistic human pathogen and the cause of biofilm-associated infections of the heart, catheterized urinary tract, wounds, and the dysbiotic gut where it can expand to high numbers upon microbiome perturbations. TheE. faecalissortase-assembled endocarditis and biofilm associated pilus (Ebp) is involved in adhesion and biofilm formationin vitroandin vivo. Extracellular electron transfer (EET) also promotesE. faecalisbiofilm formation in iron-rich environments, however neither the mechanism underlying EET nor its role in virulence was previously known. Here we show that iron associated with Ebp serve as a terminal electron acceptor for EET, leading to extracellular iron reduction and intracellular iron accumulation. We found that a MIDAS motif within the EbpA tip adhesin is required for interaction with iron, EET, and FeoB-mediated iron uptake. We demonstrate that MenB and Ndh3, essential components of the aerobic respiratory chain and a specialized flavin-mediated electron transport chain, respectively, are required for iron-mediated EET. In addition, using a mouse gastrointestinal (GI) colonization model, we show that EET is essential for colonization of the GI tract, and Ebp is essential for augmentedE. faecalisGI colonization when dietary iron is in excess. Taken together, our findings show that pilus mediated capture of iron within biofilms enables EET-mediated iron acquisition inE. faecalis, and that these processes plays an important role inE. faecalisexpansion in the GI tract.SignificanceUnderstanding enterococcal biofilm development is the first step towards improved therapeutics for the often antimicrobial resistant infections caused by these bacteria. Here we report a role forEnterococcus faecalisendocarditis and biofilm associated pili (Ebp) in mediating iron-dependent biofilm growth and contributing to extracellular electron transfer (EET) which in turn promotes iron acquisition. Furthermore, we characterize the mechanisms underlying electron transfer in theE. faecalisbiofilm. Our findings support a model in whichE. faecalisuse EET to drive the reduction of pilus-associated ferric iron, leading to iron acquisition inE. faecalisbiofilm, and contributing to enterococcal virulence in the GI tract.

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant Neisseria gonorrhoeae strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against Neisseria-specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among Neisseria spp. and that this enzyme is essential for survival of N. gonorrhoeae. DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of Neisseria DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the Neisseria n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain–domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.

Hendra virus (HeV) is a paramyxovirus that causes lethal disease in humans, for which no vaccine or antiviral agent is available. HeV V protein is central to pathogenesis through its ability to interact with cytoplasmic host proteins, playing key antiviral roles. Here we use immunoprecipitation, siRNA knockdown and confocal laser scanning microscopy to show that HeV V shuttles to and from the nucleus through specific host nuclear transporters. Spectroscopic and small angle X-ray scattering studies reveal HeV V undergoes a disorder-to-order transition upon binding to either importin α/β1 or exportin-1/Ran-GTP, dependent on the V N-terminus. Importantly, we show that specific inhibitors of nuclear transport prevent interaction with host transporters, and reduce HeV infection. These findings emphasize the critical role of host-virus interactions in HeV infection, and potential use of compounds targeting nuclear transport, such as the FDA-approved agent ivermectin, as anti-HeV agents.

The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochromecmaturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogenNeisseria meningitidis. To help understand the structural and functional aspects ofN. meningitidisDsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallographic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbDOx, n-NmDsbDRed, c-NmDsbDOxand c-NmDsbDReddiffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groupsP213,P321,P41andP1211, respectively.

Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of high-molecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.65 A resolution. The crystal lattice belonged to the hexagonal space group P6122, with unit-cell parameters a = b = 89.31, c = 290.18 A, and contained two molecules in the asymmetric unit. The crystal structure was determined by molecular replacement using a single chain of Pseudomonas aeruginosa DHDPS as the search model.

Lysine is an essential amino acid in the mammalian diet, but can be synthesised de novo in bacteria, plants and some fungi (Dogovski et al., 2009; Hutton et al., 2007). In bacteria, the lysine biosynthesis pathway, also known as the diaminopimelate (DAP) pathway (Fig. 1), yields the important metabolites meso-2,6-diaminopimelate (meso-DAP) and lysine. Lysine is utilised for protein synthesis in bacteria and forms part of the peptidoglycan cross-link structure in the cell wall of most Gram-positive species; whilst meso-DAP is the peptidoglycan cross-linking moiety in the cell wall of Gram-negative bacteria and also Gram-positive Bacillus species (Burgess et al., 2008; Mitsakos et al., 2008; Voss et al., 2010) (Fig. 1).

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.