Publications by authors named "Begoña Heras"

62 Publications

Antivirulence DsbA inhibitors attenuate serovar Typhimurium fitness without detectable resistance.

FASEB Bioadv 2021 Apr 10;3(4):231-242. Epub 2021 Feb 10.

Institute of Health and Biomedical Innovation School of Biomedical Sciences Queensland University of Technology Herston QLD Australia.

Inhibition of the DiSulfide Bond (DSB) oxidative protein folding machinery, a major facilitator of virulence in Gram-negative bacteria, represents a promising antivirulence strategy. We previously developed small molecule inhibitors of DsbA from K-12 (EcDsbA) and showed that they attenuate virulence of Gram-negative pathogens by directly inhibiting multiple diverse DsbA homologues. Here we tested the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials against serovar Typhimurium under pathophysiological conditions in vitro. We show that phenylthiophene DsbA inhibitors slow . Typhimurium growth in minimal media, phenocopying . Typhimurium isogenic null mutants. Through passaging experiments, we found that DsbA inhibitor resistance was not induced under conditions that rapidly induced resistance to ciprofloxacin, an antibiotic commonly used to treat infections. Furthermore, no mutations were identified in the gene of inhibitor-treated . Typhimurium, and . Typhimurium virulence remained susceptible to DsbA inhibitors. Our work demonstrates that under in vitro pathophysiological conditions, DsbA inhibitors can have both antivirulence and antibiotic action. Importantly, our finding that DsbA inhibitors appear to be evolutionarily robust offers promise for their further development as next-generation antimicrobials against Gram-negative pathogens.
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http://dx.doi.org/10.1096/fba.2020-00100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019255PMC
April 2021

BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Antioxid Redox Signal 2021 Apr 12. Epub 2021 Apr 12.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia.

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 termed bovine colonization factor protein H (BcfH) and defined its role in virulence. In the conserved bovine colonization factor () 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 proline instead of the conserved -proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to fimbrial biogenesis. 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.
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http://dx.doi.org/10.1089/ars.2020.8218DOI Listing
April 2021

Detection of Bacterial Membrane Vesicles by NOD-Like Receptors.

Int J Mol Sci 2021 Jan 20;22(3). Epub 2021 Jan 20.

Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora 3086, Australia.

Bacterial membrane vesicles (BMVs) are nanoparticles produced by both Gram-negative and Gram-positive bacteria that can function to modulate immunity in the host. Both outer membrane vesicles (OMVs) and membrane vesicles (MVs), which are released by Gram-negative and Gram-positive bacteria, respectively, contain cargo derived from their parent bacterium, including immune stimulating molecules such as proteins, lipids and nucleic acids. Of these, peptidoglycan (PG) and lipopolysaccharide (LPS) are able to activate host innate immune pattern recognition receptors (PRRs), known as NOD-like receptors (NLRs), such as nucleotide-binding oligomerisation domain-containing protein (NOD) 1, NOD2 and NLRP3. NLR activation is a key driver of inflammation in the host, and BMVs derived from both pathogenic and commensal bacteria have been shown to package PG and LPS in order to modulate the host immune response using NLR-dependent mechanisms. Here, we discuss the packaging of immunostimulatory cargo within OMVs and MVs, their detection by NLRs and the cytokines produced by host cells in response to their detection. Additionally, commensal derived BMVs are thought to shape immunity and contribute to homeostasis in the gut, therefore we also highlight the interactions of commensal derived BMVs with NLRs and their roles in limiting inflammatory diseases.
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http://dx.doi.org/10.3390/ijms22031005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863931PMC
January 2021

A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics.

Sci Rep 2021 Jan 15;11(1):1569. Epub 2021 Jan 15.

Institute of Health and Biomedical Innovation and Centre for Immunology and Infection Control, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, 4059, Australia.

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors.
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http://dx.doi.org/10.1038/s41598-021-81007-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810732PMC
January 2021

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiL).

J Med Chem 2020 07 24;63(13):6863-6875. Epub 2020 Jun 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00111DOI Listing
July 2020

A Molecular Chameleon for Mapping Subcellular Polarity in an Unfolded Proteome Environment.

Angew Chem Int Ed Engl 2020 06 8;59(25):10129-10135. Epub 2020 Jan 8.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.

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.
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http://dx.doi.org/10.1002/anie.201914263DOI Listing
June 2020

Molecular and structural insights into an asymmetric proteolytic complex (ClpP1P2) from Mycobacterium smegmatis.

Sci Rep 2019 12 2;9(1):18019. Epub 2019 Dec 2.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, Australia.

The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis (Msm) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of ClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.
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http://dx.doi.org/10.1038/s41598-019-53736-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889138PMC
December 2019

The Fragment-Based Development of a Benzofuran Hit as a New Class of DsbA Inhibitors.

Molecules 2019 Oct 18;24(20). Epub 2019 Oct 18.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from (DsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to DsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of DsbA. Binding affinity was calculated based on NMR studies and compounds and were identified as the highest affinity binders with dissociation constants () of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of DsbA inhibitors.
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http://dx.doi.org/10.3390/molecules24203756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832960PMC
October 2019

The Scs disulfide reductase system cooperates with the metallochaperone CueP in copper resistance.

J Biol Chem 2019 11 23;294(44):15876-15888. Epub 2019 Aug 23.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Bundoora, Victoria 3083, Australia

The human pathogen serovar Typhimurium ( 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 () gene cluster, associated with increased tolerance to copper. To better understand the function of the 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 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 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 regulon, a major copper resistance mechanism in 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.
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http://dx.doi.org/10.1074/jbc.RA119.010164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827279PMC
November 2019

Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation.

Sci Rep 2019 07 31;9(1):11100. Epub 2019 Jul 31.

Université Clermont Auvergne, INRA, UMR454 MEDiS, 63000, Clermont-Ferrand, France.

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 Ag43 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.
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http://dx.doi.org/10.1038/s41598-019-47608-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668479PMC
July 2019

Unique structural features of a bacterial autotransporter adhesin suggest mechanisms for interaction with host macromolecules.

Nat Commun 2019 04 29;10(1):1967. Epub 2019 Apr 29.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, VIC, Australia.

Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core β-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter β-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.
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http://dx.doi.org/10.1038/s41467-019-09814-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488583PMC
April 2019

CrmA orthologs from diverse poxviruses potently inhibit caspases-1 and -8, yet cleavage site mutagenesis frequently produces caspase-1-specific variants.

Biochem J 2019 05 7;476(9):1335-1357. Epub 2019 May 7.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Australia

Poxviruses encode many proteins that enable them to evade host anti-viral defense mechanisms. Spi-2 proteins, including Cowpox virus CrmA, suppress anti-viral immune responses and contribute to poxviral pathogenesis and lethality. These proteins are 'serpin' protease inhibitors, which function via a pseudosubstrate mechanism involving initial interactions between the protease and a cleavage site within the serpin. A conformational change within the serpin interrupts the cleavage reaction, deforming the protease active site and preventing dissociation. Spi-2 proteins like CrmA potently inhibit caspases-1, -4 and -5, which produce proinflammatory cytokines, and caspase-8, which facilitates cytotoxic lymphocyte-mediated target cell death. It is not clear whether both of these functions are equally perilous for the virus, or whether only one must be suppressed for poxviral infectivity and spread but the other is coincidently inhibited merely because these caspases are biochemically similar. We compared the caspase specificity of CrmA to three orthologs from orthopoxviruses and four from more distant chordopoxviruses. All potently blocked caspases-1, -4, -5 and -8 activity but exhibited negligible inhibition of caspases-2, -3 and -6. The orthologs differed markedly in their propensity to inhibit non-mammalian caspases. We determined the specificity of CrmA mutants bearing various residues in positions P4, P3 and P2 of the cleavage site. Almost all variants retained the ability to inhibit caspase-1, but many lacked caspase-8 inhibitory activity. The retention of Spi-2 proteins' caspase-8 specificity during chordopoxvirus evolution, despite this function being readily lost through cleavage site mutagenesis, suggests that caspase-8 inhibition is crucial for poxviral pathogenesis and spread.
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http://dx.doi.org/10.1042/BCJ20190202DOI Listing
May 2019

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase.

Acta Crystallogr D Struct Biol 2019 Mar 26;75(Pt 3):283-295. Epub 2019 Feb 26.

Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia.

Disulfide-bond-forming (DSB) oxidative folding enzymes are master regulators of virulence that are localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K-12 consists of a dithiol-oxidizing redox-relay pair (DsbA/B), a disulfide-isomerizing redox-relay pair (DsbC/D) and the specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast, the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these, α-DsbA1 and α-DsbB, form a redox-relay pair analogous to DsbA/B from E. coli. The third enzyme, α-DsbA2, incorporates a DsbA-like sequence but does not interact with α-DsbB. In comparison to other DsbA enzymes, α-DsbA2 has ∼50 extra N-terminal residues (excluding the signal peptide). The crystal structure of α-DsbA2ΔN, an N-terminally truncated form in which these ∼50 residues are removed, confirms the DsbA-like nature of this domain. However, α-DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. Firstly, α-DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase: i.e. its role is to shuffle rather than to introduce disulfide bonds. Moreover, small-angle X-ray scattering (SAXS) of α-DsbA2 reveals a homotrimeric arrangement that differs from those of the other characterized bacterial disulfide isomerases DsbC from Escherichia coli (homodimeric) and ScsC from Proteus mirabilis (PmScsC; homotrimeric with a shape-shifter peptide). α-DsbA2 lacks the shape-shifter motif and SAXS data suggest that it is less flexible than PmScsC. These results allow conclusions to be drawn about the factors that are required for functionally equivalent disulfide isomerase enzymatic activity across structurally diverse protein architectures.
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http://dx.doi.org/10.1107/S2059798318018442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450060PMC
March 2019

Author Correction: A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2019 03 1;10(1):976. Epub 2019 Mar 1.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD, 4072, Australia.

This Article contains errors in Fig. 1, Table 1 and the Methods section. In panel c, the labels for PmScsC and EcDsbC in the upper two curves are interchanged. In Table 1 and the Methods section entitled 'Extended structure', the space group of the extended PmScsC structure is incorrectly referred to as H3 and should read H32. Correct versions of Fig. 1 and Table 1 are presented below; the errors have not been corrected in the Article.
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http://dx.doi.org/10.1038/s41467-019-08920-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397206PMC
March 2019

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens.

J Biol Chem 2018 10 4;293(43):16559-16571. Epub 2018 Sep 4.

From the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Victoria, Australia,

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant 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 -specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among spp. and that this enzyme is essential for survival of 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 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 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.
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http://dx.doi.org/10.1074/jbc.RA118.004847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204915PMC
October 2018

Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis.

Acta Crystallogr F Struct Biol Commun 2018 01 1;74(Pt 1):31-38. Epub 2018 Jan 1.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia.

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, cytochrome c maturation 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 pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, 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-NmDsbD, n-NmDsbD, c-NmDsbD and c-NmDsbD diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P23, P321, P4 and P121, respectively.
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http://dx.doi.org/10.1107/S2053230X17017800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947690PMC
January 2018

Inhibition of Diverse DsbA Enzymes in Multi-DsbA Encoding Pathogens.

Antioxid Redox Signal 2018 09 1;29(7):653-666. Epub 2018 Feb 1.

2 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora, Australia .

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.
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http://dx.doi.org/10.1089/ars.2017.7104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067686PMC
September 2018

A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2017 07 19;8:16065. Epub 2017 Jul 19.

Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.

Copper resistance is a key virulence trait of the uropathogen Proteus mirabilis. Here we show that P. mirabilis ScsC (PmScsC) contributes to this defence mechanism by enabling swarming in the presence of copper. We also demonstrate that PmScsC is a thioredoxin-like disulfide isomerase but, unlike other characterized proteins in this family, it is trimeric. PmScsC trimerization and its active site cysteine are required for wild-type swarming activity in the presence of copper. Moreover, PmScsC exhibits unprecedented motion as a consequence of a shape-shifting motif linking the catalytic and trimerization domains. The linker accesses strand, loop and helical conformations enabling the sampling of an enormous folding landscape by the catalytic domains. Mutation of the shape-shifting motif abolishes disulfide isomerase activity, as does removal of the trimerization domain, showing that both features are essential to foldase function. More broadly, the shape-shifter peptide has the potential for 'plug and play' application in protein engineering.
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http://dx.doi.org/10.1038/ncomms16065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524982PMC
July 2017

Autotransporter Adhesins in Escherichia coli Pathogenesis.

Proteomics 2017 Dec 12;17(23-24). Epub 2017 Oct 12.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

Most bacteria produce adhesion molecules to facilitate the interaction with host cells and establish successful infections. An important group of bacterial adhesins belong to the autotransporter (AT) superfamily, the largest group of secreted and outer membrane proteins in Gram-negative bacteria. AT adhesins possess diverse functions that facilitate bacterial colonisation, survival and persistence, and as such are often associated with increased bacterial fitness and pathogenic potential. In this review, we will describe AIDA-I type AT adhesins, which comprise the biggest and most diverse group in the AT family. We will focus on Escherichia coli proteins and define general aspects of their biogenesis, distribution, structural properties and key roles in infection.
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http://dx.doi.org/10.1002/pmic.201600431DOI Listing
December 2017

H, N, C and C assignments of the two periplasmic domains of Neisseria meningitidis DsbD.

Biomol NMR Assign 2017 Oct 6;11(2):181-186. Epub 2017 Jun 6.

La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia.

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H, N, C and C) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using C shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.
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http://dx.doi.org/10.1007/s12104-017-9743-xDOI Listing
October 2017

Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase.

PLoS One 2016 28;11(12):e0168485. Epub 2016 Dec 28.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, Australia.

The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168485PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5193440PMC
July 2017

Bioinformatics Tools and Resources for Analyzing Protein Structures.

Methods Mol Biol 2017 ;1549:209-220

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Rm 521, LIMS1, Kingsbury Drive, Bundoora, Melbourne, VIC, 3086, Australia.

The dramatic increase in the number of protein sequences and structures deposited in biological databases has led to the development of many bioinformatics tools and programs to manage, validate, compare, and interpret this large volume of data. In addition, powerful tools are being developed to use this sequence and structural data to facilitate protein classification and infer biological function of newly identified proteins. This chapter covers freely available bioinformatics resources on the World Wide Web that are commonly used for protein structure analysis.
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http://dx.doi.org/10.1007/978-1-4939-6740-7_16DOI Listing
February 2018

Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

Molecules 2016 Jul 16;21(7). Epub 2016 Jul 16.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Bundoora, Vic 3083, Australia.

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.
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http://dx.doi.org/10.3390/molecules21070811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273893PMC
July 2016

Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein.

mBio 2016 Mar 15;7(2):e02046. Epub 2016 Mar 15.

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Unlabelled: 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.
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http://dx.doi.org/10.1128/mBio.02046-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807369PMC
March 2016

Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility.

Acta Crystallogr D Biol Crystallogr 2015 Dec 26;71(Pt 12):2386-95. Epub 2015 Nov 26.

Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072, Australia.

Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.
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http://dx.doi.org/10.1107/S1399004715018519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667283PMC
December 2015

Lenvatinib versus placebo in radioiodine-refractory thyroid cancer.

N Engl J Med 2015 Feb;372(7):621-30

The authors' affiliations are listed in the Appendix.

Background: Lenvatinib, an oral inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, fibroblast growth factor receptors 1 through 4, platelet-derived growth factor receptor α, RET, and KIT, showed clinical activity in a phase 2 study involving patients with differentiated thyroid cancer that was refractory to radioiodine (iodine-131).

Methods: In our phase 3, randomized, double-blind, multicenter study involving patients with progressive thyroid cancer that was refractory to iodine-131, we randomly assigned 261 patients to receive lenvatinib (at a daily dose of 24 mg per day in 28-day cycles) and 131 patients to receive placebo. At the time of disease progression, patients in the placebo group could receive open-label lenvatinib. The primary end point was progression-free survival. Secondary end points included the response rate, overall survival, and safety.

Results: The median progression-free survival was 18.3 months in the lenvatinib group and 3.6 months in the placebo group (hazard ratio for progression or death, 0.21; 99% confidence interval, 0.14 to 0.31; P<0.001). A progression-free survival benefit associated with lenvatinib was observed in all prespecified subgroups. The response rate was 64.8% in the lenvatinib group (4 complete responses and 165 partial responses) and 1.5% in the placebo group (P<0.001). The median overall survival was not reached in either group. Treatment-related adverse effects of any grade, which occurred in more than 40% of patients in the lenvatinib group, were hypertension (in 67.8% of the patients), diarrhea (in 59.4%), fatigue or asthenia (in 59.0%), decreased appetite (in 50.2%), decreased weight (in 46.4%), and nausea (in 41.0%). Discontinuations of the study drug because of adverse effects occurred in 37 patients who received lenvatinib (14.2%) and 3 patients who received placebo (2.3%). In the lenvatinib group, 6 of 20 deaths that occurred during the treatment period were considered to be drug-related.

Conclusions: Lenvatinib, as compared with placebo, was associated with significant improvements in progression-free survival and the response rate among patients with iodine-131-refractory thyroid cancer. Patients who received lenvatinib had more adverse effects. (Funded by Eisai; SELECT ClinicalTrials.gov number, NCT01321554.).
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http://dx.doi.org/10.1056/NEJMoa1406470DOI Listing
February 2015

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

Angew Chem Int Ed Engl 2015 Feb 30;54(7):2179-84. Epub 2014 Dec 30.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052 (Australia) http://www.pharm.monash.edu.au.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.
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http://dx.doi.org/10.1002/anie.201410341DOI Listing
February 2015

Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to elongation factor EF-Tu reveals a novel protein interaction site.

J Biol Chem 2014 Jul 23;289(29):19869-80. Epub 2014 May 23.

From the Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, University of Queensland, St. Lucia, Queensland 4067, Australia

The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.
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http://dx.doi.org/10.1074/jbc.M114.571737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106308PMC
July 2014

Targeting virulence not viability in the search for future antibacterials.

Br J Clin Pharmacol 2015 Feb;79(2):208-15

La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Vic.

New antibacterials need new approaches to overcome the problem of rapid antibiotic resistance. Here we review the development of potential new antibacterial drugs that do not kill bacteria or inhibit their growth, but combat disease instead by targeting bacterial virulence.
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http://dx.doi.org/10.1111/bcp.12356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309627PMC
February 2015

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping.

Proc Natl Acad Sci U S A 2014 Jan 13;111(1):457-62. Epub 2013 Dec 13.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure-function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular "Velcro-like" mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.
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http://dx.doi.org/10.1073/pnas.1311592111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890832PMC
January 2014