Mads Gabrielsen

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence blocking compounds

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

Structural and biochemical characterization of a mitochondrial peroxiredoxin from Plasmodium falciparum

Plasmodium falciparum possesses a single mitochondrion with a functional electron transport chain. During respiration, reactive oxygen species are generated that need to be removed to protect the organelle from oxidative damage. In the absence of catalase and glutathione peroxidase, the parasites rely primarily on peroxiredoxin‐linked systems for protection. We have analysed the biochemical and structural features of the mitochondrial peroxiredoxin and thioredoxin of P. falciparum. The mitochondrial localization of both proteins was confirmed by expressing green fluorescent protein fusions in parasite erythrocytic stages. Recombinant protein was kinetically characterized using the cytosolic and the mitochondrial thioredoxin (PfTrx1 and PfTrx2 respectively). The peroxiredoxin clearly preferred PfTrx2 to PfTrx1 as a reducing partner, reflected by the KM values of 11.6 μM and 130.4 μM respectively. Substitution of the two dyads asparagine‐62/tyrosine‐63 and phenylalanine‐139/alanine‐140 residues by aspartate‐phenylalaine and valine‐serine, respectively, reduced the KM for Trx1 but had no effect on the KM of Trx2 suggesting some role for these residues in the discrimination between the two substrates. Solution studies suggest that the protein exists primarily in a homodecameric form. The crystal structure of the mitochondrial peroxiredoxin reveals a fold typical of the 2‐Cys class peroxiredoxins and a dimeric form with an intermolecular disulphide bridge between Cys67 and Cys187. These results show that the mitochondrial peroxiredoxin of P. falciparum occurs in both dimeric and decameric forms when purified under non‐reducing conditions.

Peripheral Complexes of Purple Bacteria

This chapter uses the LH2 complex from Rhodopseudomonas acidophila strain 10050 as an example to describe the current understanding of the structure of purple bacterial peripheral antenna complexes. It summarizes both what is and what is not understood. So far the structures of ‘standard’ LH2 complexes, such as those from Rhodopseudomonas acidophila, are rather well characterized. In contrast, there is a dearth of structural information on complexes with more unusual spectroscopic properties. There is also very little information available on how these antenna complexes are assembled.

Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

The crystal structure of a plant 2C‐methyl‐D‐erythritol 4‐phosphate cytidylyltransferase exhibits a distinct quaternary structure compared to bacterial homologues and a possible role in feedback regulation for cytidine monophosphate

The homodimeric 2C‐methyl‐d‐erythritol 4‐phosphate cytidylyltransferase contributes to the nonmevalonate pathway of isoprenoid biosynthesis. The crystal structure of the catalytic domain of the recombinant enzyme derived from the plant Arabidopsis thaliana has been solved by molecular replacement and refined to 2.0 Å resolution. The structure contains cytidine monophosphate bound in the active site, a ligand that has been acquired from the bacterial expression system, and this observation suggests a mechanism for feedback regulation of enzyme activity. Comparisons with bacterial enzyme structures, in particular the enzyme from Escherichia coli, indicate that whilst individual subunits overlay well, the arrangement of subunits in each functional dimer is different. That distinct quaternary structures are available, in conjunction with the observation that the protein structure contains localized areas of disorder, suggests that conformational flexibility may contribute to the function of this enzyme.

Polarized Cell Motility Induces Hydrogen Peroxide to Inhibit Cofilin via Cysteine Oxidation

Summary Mesenchymal cell motility is driven by polarized actin polymerization [1]. Signals at the leading edge recruit actin polymerization machinery to promote membrane protrusion, while matrix adhesion generates tractive force to propel forward movement. To work effectively, cell motility is regulated by a complex network of signaling events that affect protein activity and localization. H2O2 has an important role as a diffusible second messenger [2], and mediates its effects through oxidation of cysteine thiols. One cell activity influenced by H2O2 is motility [3]. However, a lack of sensitive and H2O2-specific probes for measurements in live cells has not allowed for direct observation of H2O2 accumulation in migrating cells or protrusions. In addition, the identities of proteins oxidized by H2O2 that contribute to actin dynamics and cell motility have not been characterized. We now show, as determined by fluorescence lifetime imaging microscopy, that motile cells generate H2O2 at membranes and cell protrusions and that H2O2 inhibits cofilin activity through oxidation of cysteines 139 (C139) and 147 (C147). Molecular modeling suggests that C139 oxidation would sterically hinder actin association, while the increased negative charge of oxidized C147 would lead to electrostatic repulsion of the opposite negatively charged surface. Expression of oxidation-resistant cofilin impairs cell spreading, adhesion, and directional migration. These findings indicate that H2O2 production contributes to polarized cell motility through localized cofilin inhibition and that there are additional proteins oxidized during cell migration that might have similar roles.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to “sense” levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

BackgroundCucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology.FindingsCucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation.ConclusionsThe implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.

Spectral Diffusion and Electron-Phonon Coupling of the B800 BChl a Molecules in LH2 Complexes from Three Different Species of Purple Bacteria

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

Structural Characterisation of Tpx from Yersinia Pseudotuberculosis Reveals Insights Into the Binding of Salicylidene Acylhydrazide Compounds.

Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for “forced” reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds.