Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis.

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

A bacterial sulfoglycosidase highlights mucin O-glycan breakdown in the gut ecosystem.

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.

cropCSM: designing safe and potent herbicides with graph-based signatures.

Herbicides have revolutionised weed management, increased crop yields and improved profitability allowing for an increase in worldwide food security. Their widespread use, however, has also led to a rise in resistance and concerns about their environmental impact. Despite the need for potent and safe herbicidal molecules, no herbicide with a new mode of action has reached the market in 30 years. Although development of computational approaches has proven invaluable to guide rational drug discovery pipelines, leading to higher hit rates and lower attrition due to poor toxicity, little has been done in contrast for herbicide design. To fill this gap, we have developed cropCSM, a computational platform to help identify new, potent, nontoxic and environmentally safe herbicides. By using a knowledge-based approach, we identified physicochemical properties and substructures enriched in safe herbicides. By representing the small molecules as a graph, we leveraged these insights to guide the development of predictive models trained and tested on the largest collected data set of molecules with experimentally characterised herbicidal profiles to date (over 4500 compounds). In addition, we developed six new environmental and human toxicity predictors, spanning five different species to assist in molecule prioritisation. cropCSM was able to correctly identify 97% of herbicides currently available commercially, while predicting toxicity profiles with accuracies of up to 92%. We believe cropCSM will be an essential tool for the enrichment of screening libraries and to guide the development of potent and safe herbicides. We have made the method freely available through a user-friendly webserver at http://biosig.unimelb.edu.au/crop_csm.

Novel small molecules that increase the susceptibility of Neisseria gonorrhoeae to cationic antimicrobial peptides by inhibiting lipid A phosphoethanolamine transferase.

OBJECTIVES: Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosphoethanolamine to lipid A, results in attenuation of the pathogen in infection models. Small molecules that inhibit EptA are predicted to enhance natural clearance of gonococci via the human innate immune response. METHODS: A library of small-fragment compounds was tested for the ability to enhance susceptibility of the reference strain N. gonorrhoeae FA1090 to polymyxin B. The effect of these compounds on lipid A synthesis and viability in models of infection were tested. RESULTS: Three compounds, 135, 136 and 137, enhanced susceptibility of strain FA1090 to polymyxin B by 4-fold. Pre-treatment of bacterial cells with all three compounds resulted in enhanced killing by macrophages. Only lipid A from bacterial cells exposed to compound 137 showed a 17% reduction in the level of decoration of lipid A with phosphoethanolamine by MALDI-TOF MS analysis and reduced stimulation of cytokine responses in THP-1 cells. Binding of 137 occurred with higher affinity to purified EptA than the starting material, as determined by 1D saturation transfer difference NMR. Treatment of eight MDR strains with 137 increased susceptibility to polymyxin B in all cases. CONCLUSIONS: Small molecules have been designed that bind to EptA, inhibit addition of phosphoethanolamine to lipid A and can sensitize N. gonorrhoeae to killing by macrophages.

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.

Red Algal Molecules - Synthesis of Methyl Neo-ß-carrabioside and Its S-Linked Variant via Two Synthetic Routes: A Late Stage Ring Closure and Using a 3,6-Anhydro-d-galactosyl Donor.

Methyl neo-ß-carrabioside has been synthesized for the first time, employing either a late stage ring closure to install the required 3,6-anhydro-bridge or a suitable 3,6-anhydro-galactosyl donor to form the unfavored 1,2-cis-equatorial α-linkage. Using the late stage ring closure approach, an S-linked analogue of methyl neo-ß-carrabioside was also realized. These compounds have applications in the identification and characterization of marine bacterial exo-α-3,6-anhydro-d-galactosidases that have specific activity on red algal neo-carrageenan oligosaccharides, such as those found in both family 127 and 129 of the glycoside hydrolases. In addition a biochemical assay using the synthesized methyl neo-ß-carrabioside and the marine bacterial exo-α-3,6-anhydro-d-galactosidase ZgGH129 demonstrates that the minimum substrate unit for the enzyme is neo-ß-carrabiose.

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding.

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.

Targeting plant DIHYDROFOLATE REDUCTASE with antifolates and mechanisms for genetic resistance.

The folate biosynthetic pathway and its key enzyme dihydrofolate reductase (DHFR) is a popular target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. Despite its importance, little is known about plant DHFRs, which, like the enzymes from the malarial parasite Plasmodium, are bifunctional, possessing DHFR and thymidylate synthase (TS) domains. Here using genetic knockout lines we confirmed that either DHFR-TS1 or DHFR-TS2 (but not DHFR-TS3) was essential for seed development. Screening mutated Arabidopsis thaliana seeds for resistance to antimalarial DHFR-inhibitor drugs pyrimethamine and cycloguanil identified causal lesions in DHFR-TS1 and DHFR-TS2, respectively, near the predicted substrate-binding site. The different drug resistance profiles for the plants, enabled by the G137D mutation in DHFR-TS1 and the A71V mutation in DHFR-TS2, were consistent with biochemical studies using recombinant proteins and could be explained by structural models. These findings provide a great improvement in our understanding of plant DHFR-TS and suggest how plant-specific inhibitors might be developed, as DHFR is not currently targeted by commercial herbicides.

Synthetic and Crystallographic Insight into Exploiting sp2 Hybridization in the Development of α-l-Fucosidase Inhibitors.

The sugar fucose plays a myriad of roles in biological recognition. Enzymes hydrolyzing fucose from glycoconjugates, α-l-fucosidases, are important targets for inhibitor and probe development. Here we describe the synthesis and evaluation of novel α-l-fucosidase inhibitors, with X-ray crystallographic analysis using an α-l-fucosidase from Bacteroides thetaiotamicron helping to lay a foundation for future development of inhibitors for this important enzyme class.

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.

Biosynthesis of a Tricyclo[6.2.2.02,7 ]dodecane System by a Berberine Bridge Enzyme-Like Aldolase.

The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.02,7 ]dodecane skeleton in the post-assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.

The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors.

The α-N-acetylgalactosaminidase from the probiotic bacterium Bifidobacterium bifidum (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAcα1-Ser/Thr). NagBb is involved in assimilation of O-glycans on mucin glycoproteins by B. bifidum in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca2+, is involved in the recognition of the GalNAc N-acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-α-N-acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.

Angled Vortex Fluidic Mediated Multicomponent Photocatalytic and Transition Metal-Catalyzed Reactions.

The dynamic thin film formed in an angled rapidly rotating tube in a vortex fluidic device (VFD) is effective in facilitating multicomponent reactions (MCRs) as photocatalytic or metal-mediated processes. Here, we demonstrate the utility of the VFD by using two known MCRs, an Ugi-type three component reaction and an A3 -coupling reaction. The Ugi-type reaction can be done in either confined or continuous-flow modes of operation of the microfluidic platform whereas the A3 -coupling reaction was optimized for the confined mode of operation. The examples tested gave excellent yields with short reaction times.

Integrating thin film microfluidics in developing a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases.

A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a potent inhibitor of α-N-acetylhexosaminidases, DGJNAc, has been developed.

Exploiting sp2 -Hybridisation in the Development of Potent 1,5-α-l-Arabinanase Inhibitors.

The synthesis of potent inhibitors of GH93 arabinanases as well as a synthesis of a chromogenic substrate to measure GH93 arabinanase activity are described. An insight into the reasons behind the potency of the inhibitors was gained through X-ray crystallographic analysis of the arabinanase Arb93A from Fusarium graminearum. These compounds lay a foundation for future inhibitor development as well as for the use of the chromogenic substrate in biochemical studies of GH93 arabinanases.

Synergistic activity of fosfomycin, ß-lactams and peptidoglycan recycling inhibition against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and ß-lactams in Pseudomonas aeruginosa METHODS: Fosfomycin MICs were determined by broth microdilution and Etest for a panel of 47 PAO1 mutants defective in several components of PG recycling and/or AmpC induction pathways. PAO1 fosfomycin MICs were also determined in the presence of a 5 mM concentration of the NagZ inhibitor PUGNAc. Population analysis of fosfomycin susceptibility and characterization of the resistant mutants that emerged was also performed for selected strains. Finally, fosfomycin, imipenem and fosfomycin + imipenem killing curves were assessed. RESULTS: Mutants defective in AmpG, NagZ or all three AmpD amidases showed a marked increase in fosfomycin susceptibility (at least two 2-fold dilutions with respect to WT PAO1). Moreover, PAO1 fosfomycin MICs were consistently reduced from 48 to 24 mg/L in the presence of a 5 mM concentration of PUGNAc. Fosfomycin hypersusceptibility of the ampG, nagZ and triple ampD mutants was also clearly confirmed in the performed population analysis, although the emergence of resistant mutants, through GlpT mutations, was not avoided. Synergy between fosfomycin and imipenem was evidenced for the WT strain, the AmpC-hyperproducing strain (triple AmpD mutant) and the NagZ and AmpG mutants in killing curves. Moreover, regrowth of resistant mutants was not evidenced for the combination. CONCLUSIONS: PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with ß-lactams and fosfomycin and therefore further development of these molecules is encouraged.

Vortex Fluidic Chemical Transformations.

Driving chemical transformations in dynamic thin films represents a rapidly thriving and diversifying research area. Dynamic thin films provide a number of benefits including large surface areas, high shearing rates, rapid heat and mass transfer, micromixing and fluidic pressure waves. Combinations of these effects provide an avant-garde style of conducting chemical reactions with surprising and unusual outcomes. The vortex fluidic device (VFD) has proved its capabilities in accelerating and increasing the efficiencies of numerous organic, materials and biochemical reactions. This Minireview surveys transformations that have benefited from VFD-mediated processing, and identifies concepts driving the effectiveness of vortex-based dynamic thin films.

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

BACKGROUND: Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. RESULTS: Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, ß-elemene, ß-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 µg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 µg/ml). CONCLUSIONS: This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.

Exploiting the Evolutionary Relationship between Malarial Parasites and Plants To Develop New Herbicides.

Herbicide resistance is driving a need to develop new herbicides. The evolutionary relationship between apicomplexan parasites, such as those causing malaria, and plants is close enough that many antimalarial drugs are herbicidal and so represent novel scaffolds for herbicide development. Using a compound library from the Medicines for Malaria Venture, the model plant Arabidopsis thaliana, and a physicochemical database of known herbicides, a compound was discovered that showed post-emergence herbicidal activity equal to commercial herbicides. Using structure-activity analysis, important points for its potency were found. The compound was also tested and found to be active against common crop weeds. Physiological profiling suggested the compound was a photosystem II inhibitor, representing a new scaffold for herbicide development. Overall this approach demonstrates the viability of using antimalarial compounds as lead compounds for the development of much needed new herbicides.