Emily Furlong

Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo

AIMS The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacteriums resistance to multiple antibiotics. In a number of clinically relevant Gram-negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalyzing the formation of disulfide bonds in secreted and membrane-associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. RESULTS Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A dsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a BALB/c mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 Å resolution crystal structure of BpsDsbA revealed differences from the classic member of this family Escherichia coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating that the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. INNOVATION This study has characterized BpsDsbA biochemically and structurally and determined that it is required for virulence of B. pseudomallei. CONCLUSION These data establish a critical role for BpsDsbA in B. pseudomallei infection, which in combination with our structural characterization of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug-resistant organism.

Disulfide isomerase activity of the dynamic, trimeric Proteus mirabilis ScsC protein is primed by the tandem immunoglobulin-fold domain of ScsB.

Correct disulfide bond formation is essential for proper folding of many proteins, including bacterial virulence factors. The suppressor of copper sensitivity (Scs) proteins have roles in dithiol/disulfide interchange and the bacterial response to copper stress. Encoded in a four-gene cassette (ScsABCD) present in many Gram-negative bacteria, the Scs proteins are enigmatic and poorly characterized. Here, we show that the periplasmic α-domain of the membrane protein ScsB in the Gram-negative bacterium Proteus mirabilis forms a redox relay with the soluble periplasmic protein PmScsC. We also found that the periplasmic α-domain is sufficient to activate the disulfide isomerase activity of PmScsC. The crystal structure of PmScsBα at a resolution of 1.54 Å revealed that it comprises two structurally similar immunoglobulin-like folds, one of which includes a putative redox-active site with the sequence CXXXC. We confirmed the importance of these cysteine residues for PmScsBα function, and in addition, we engineered cysteine variants that produced a stable complex between PmScsC and PmScsBα. Using small-angle X-ray and neutron scattering analyses with contrast variation, we determined a low-resolution structure of the PmScsC–PmScsBα complex. The structural model of this complex suggested that PmScsBα uses both of its immunoglobulin-like folds to interact with PmScsC and revealed that the highly dynamic PmScsC becomes ordered upon PmScsBα binding. These findings add to our understanding of the poorly characterized Scs proteins.

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

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.

Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase, PmScsC

Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance – a key virulence trait of the uropathogen. Each protomer of the enzyme has an N-terminal trimerisation stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, we characterise two PmScsC variants, PmScsCΔN and PmScsCΔLinker. PmScsCΔN, is an N-terminally truncated form of the protomer with two helices of the trimerisation stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsCΔN reported here reveals – as expected – a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant PmScsCΔLinker was designed to remove the 11 amino acid linker and we show that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsCΔLinker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentration, whereas PmScsCΔLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase our understanding of disulfide isomerase activity, showing how (i) oligomerisation, (ii) spacing between, and (iii) dynamic motion of, catalytic domains in PmScsC all contribute to its native function.

Antifungal Benzo[b]thiophene 1,1-dioxide IMPDH Inhibitors Exhibit Pan-Assay Interference (PAINS) Profiles

Fungi cause serious life-threatening infections in immunocompromised individuals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor.

Understanding IMPDH and ADSS in Cryptococcus neoformans and their inhibitor design

Infections caused by fungi and bacteria are a major global problem. In humans, cryptococcosis is one of the life-threatening infection caused by fungi Cryptococcus neoformans. The treatment is based on three antifungal agents: Amphotericin B, Fluconazole and Flucytosine. However, there are significant side effects associated with these drugs and increased drug resistance in some cases. The discovery of new antifungals is essential to fight against the rise of invasive infections caused by fungal pathogens. In all organisms, purine metabolic pathway is essential for the biosynthesis of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) nucleotides. In de novo purine biosynthetic pathway, Inosine monophosphate (IMP) acts as a branch point substrate for the formation of ATP and GTP through two different directional pathways catalyzed by Adenylosuccinate synthetase (ADSS) and Inosine monophosphate dehydrogense (IMPDH). To design inhibitors of C. neoformans IMPDH (CnIMPDH) the high-throughput screening was conducted with 114,000 drug-like compounds from Walter and Eliza Hall Compound Collection before to onset of my thesis work. One of the interesting hit set that I have been studying is based on benzothiophene-1,1-dioxide moiety and by modification of aromatic substituents on this hit molecule we found potent CnIMPDH inhibitors with antifungal activity. The studies for identifying the binding site of these inhibitors through kinetics and crystallography are under progress and will be published soon. Regarding C. neoformans ADSS (CnADSS), the structural elements essential for the substrate (IMP) and cofactor (GTP) binding are studied using protein crystallography technique. This information could be useful for the design of inhibitors through computational studies. The plan of utilizing computational studies is to substitute the traditional high throughput screening (HTS) that requires lot of time and cost for identification of hits and their development to lead molecules. The in vitro assays on identified hits for inhibition of CnADSS (IC50) and finding the site of binding through crystallography are in progress.

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

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.