Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence ...factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B. pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B. pseudomallei virulence factors identified by both methods are homologues of PenI family β-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.
What makes politically dissatisfied citizens enthusiastic about deliberation? And what makes them hate it instead? Based on a picture task embedded in a series of focus groups conducted in Belgium, ...we argue that differences in sense-making help to explain why dissatisfied citizens (do not) support deliberation. We focus on two groups of dissatisfied citizens: non-partisan activists and politically disadvantaged citizens. For both groups, we find that when they thought of deliberation as low-key, informal discussion, they linked it to respectful communication and beneficial outcomes; when they thought of it as formalized, structured discussion, their appraisals became much more negative. For researchers of deliberation, our results make clear that we should be careful in asking citizens what they think about ‘deliberation’ without inquiring into the way they interpret it. For deliberation practitioners, our findings underline the relevance of integrating informal interactions into the design of deliberative institutions.
Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary ...body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and-using recombinant purified proteins-demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein's thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB differentiation coincided with the oxidation of the chlamydial outer membrane complex (COMC). Together with our demonstration of an active redox pairing, our findings suggest a potential role for CtDsbA and CtDsbB in the critical disulfide bond formation step in the highly regulated development cycle.
Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have ...isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.
Suppressor of copper sensitivity (Scs) proteins play a role in the bacterial response to copper stress in many Gram-negative bacteria, including in the human pathogen Proteus mirabilis. Recently, the ...ScsC protein from P. mirabilis (PmScsC) was characterized as a trimeric protein with isomerase activity that contributes to the ability of the bacterium to swarm in the presence of copper. The CXXC motif catalytic cysteines of PmScsC are maintained in their active reduced state by the action of its membrane-bound partner protein, the Proteus mirabilis ScsB (PmScsB). Thus, PmScsC and PmScsB form a redox relay in vivo. The predicted domain arrangement of PmScsB comprises a central transmembrane β-domain and two soluble, periplasmic domains, the N-terminal α-domain and C-terminal γ-domain. Here, we provide a procedure for the recombinant expression and purification of the full-length PmScsB protein. Using Lemo21 (DE3) cells we expressed PmScsB and, after extraction and purification, we were able to achieve a yield of 3 mg of purified protein per 8 L of bacterial culture. Furthermore, using two orthogonal methods - AMS labelling of free thiols and a scrambled RNase A activity assay - PmScsB is shown to catalyze the reduction of PmScsC. Our results demonstrate that the PmScsC and PmScsB redox relay can be reconstituted in vitro using recombinant full-length PmScsB membrane protein. This finding provides a promising starting point for the in vitro biochemical and structural characterization of the P. mirabilis ScsC and ScsB interaction.
•The bacterial membrane protein ScsB was purified using an E. coli expression system.•ScsB contains two periplasmic domains (α- and γ-) connected by a transmembrane β-domain.•ScsB forms a redox relay with its partner protein ScsC to mediate disulfide isomerization.
The introduction of disulfide bonds into periplasmic proteins is a critical process in many Gram‐negative bacteria. The formation and regulation of protein disulfide bonds have been linked to the ...production of virulence factors. Understanding the different pathways involved in this process is important in the development of strategies to disarm pathogenic bacteria. The well characterized disulfide bond‐forming (DSB) proteins play a key role by introducing or isomerizing disulfide bonds between cysteines in substrate proteins. Curiously, the suppressor of copper sensitivity C proteins (ScsCs), which are part of the bacterial copper‐resistance response, share structural and functional similarities with DSB oxidase and isomerase proteins, including the presence of a catalytic thioredoxin domain. However, the oxidoreductase activity of ScsC varies with its oligomerization state, which depends on a poorly conserved N‐terminal domain. Here, the structure and function of Caulobacter crescentus ScsC (CcScsC) have been characterized. It is shown that CcScsC binds copper in the copper(I) form with subpicomolar affinity and that its isomerase activity is comparable to that of Escherichia coli DsbC, the prototypical dimeric bacterial isomerase. It is also reported that CcScsC functionally complements trimeric Proteus mirabilis ScsC (PmScsC) in vivo, enabling the swarming of P. mirabilis in the presence of copper. Using mass photometry and small‐angle X‐ray scattering (SAXS) the protein is demonstrated to be trimeric in solution, like PmScsC, and not dimeric like EcDsbC. The crystal structure of CcScsC was also determined at a resolution of 2.6 Å, confirming the trimeric state and indicating that the trimerization results from interactions between the N‐terminal α‐helical domains of three CcScsC protomers. The SAXS data analysis suggested that the protomers are dynamic, like those of PmScsC, and are able to sample different conformations in solution.
The characterization of the suppressor of copper sensitivity protein C from C. crescentus is reported.
Disulfide‐bond‐forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram‐negative bacteria. Disulfide‐bond‐forming protein A (DsbA) catalyzes the formation of the disulfide bonds ...necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide (1) and 4‐methoxy‐N‐phenylbenzenesulfonamide (2), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co‐crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side‐chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D‐NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 mM fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 mM fragment 2. Although binding was detected by both X‐ray crystallography and NMR, the binding affinity (Kd) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.
The binding properties of two drug‐like fragments to a conformationally dynamic site in disulfide bond‐forming protein A from B. pseudomallei are described.
We propose a novel neural probe which combines microfluidic channels with recording and stimulation electrodes. The developed microfabrication approach enables the concentration of every active ...element such as electrodes and the sampling inlet in close proximity on the same surface. As a first approach, full functional validation is presented in this work (in vivo testing will be presented in the next study). Electrical characterization by impedance spectroscopy is performed in order to assess the electrode properties. An advanced experimental setup enabling the validation of the fluidic functions of the neural probe is also presented. It allowed the achievement of a high temporal resolution (170 ms) during sampling as a result of the integration of a T-junction droplet generator inside the probe. The droplets reached a volume of 0.84 nL and are separated by a non-aqueous phase (perfluoromethyldecalin, PFD). This probe represents an innovative tool for neuroscientists as it can be implanted in precise brain structures while combining electrical stimulation with sampling at a high temporal resolution.
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