Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

N-acetyl-ß-hexosaminidase activity is important for chitooligosaccharide metabolism and biofilm formation in Burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic Gram-negative bacillus that can cause the disease melioidosis. Although B. pseudomallei is a recognised member of terrestrial soil microbiomes, little is known about its contribution to the saprophytic degradation of polysaccharides within its niche. For example, while chitin is predicted to be abundant within terrestrial soils the chitinolytic capacity of B. pseudomallei is yet to be defined. This study identifies and characterises a putative glycoside hydrolase, bpsl0500, which is expressed by B. pseudomallei K96243. Recombinant BPSL0500 was found to exhibit activity against substrate analogues and GlcNAc disaccharides relevant to chitinolytic N-acetyl-ß-d-hexosaminidases. In B. pseudomallei, bpsl0500 was found to be essential for both N-acetyl-ß-d-hexosaminidase activity and chitooligosaccharide metabolism. Furthermore, bpsl0500 was also observed to significantly affect biofilm deposition. These observations led to the identification of BPSL0500 activity against model disaccharide linkages that are present in biofilm exopolysaccharides, a feature that has not yet been described for chitinolytic enzymes. The results in this study indicate that chitinolytic N-acetyl-ß-d-hexosaminidases like bpsl0500 may facilitate biofilm disruption as well as chitin assimilation, providing dual functionality for saprophytic bacteria such as B. pseudomallei within the competitive soil microbiome.

Phosphoethanolamine Transferases as Drug Discovery Targets for Therapeutic Treatment of Multi-Drug Resistant Pathogenic Gram-Negative Bacteria.

Antibiotic resistance caused by multidrug-resistant (MDR) bacteria is a major challenge to global public health. Polymyxins are increasingly being used as last-in-line antibiotics to treat MDR Gram-negative bacterial infections, but resistance development renders them ineffective for empirical therapy. The main mechanism that bacteria use to defend against polymyxins is to modify the lipid A headgroups of the outer membrane by adding phosphoethanolamine (PEA) moieties. In addition to lipid A modifying PEA transferases, Gram-negative bacteria possess PEA transferases that decorate proteins and glycans. This review provides a comprehensive overview of the function, structure, and mechanism of action of PEA transferases identified in pathogenic Gram-negative bacteria. It also summarizes the current drug development progress targeting this enzyme family, which could reverse antibiotic resistance to polymyxins to restore their utility in empiric therapy.

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.

Use of Minimal Residual Disease Status to Reduce Uncertainty in Estimating Long-term Survival Outcomes for Newly Diagnosed Multiple Myeloma Patients.

Background: Demonstrating the cost-effectiveness of new treatments for multiple myeloma (MM) often relies on the extrapolation of overall survival (OS) trial data. This method can introduce uncertainty in long-term survival estimates if OS data are immature, as is often the case in newly diagnosed MM (NDMM). We explore the use of the relationship between minimal residual disease (MRD) status and OS to reduce uncertainty of long-term survival outcomes. Objectives: To evaluate if uncertainty in long-term modeled outcomes in NDMM is reduced using a response-based partitioned survival model (PSM), whereby patients were categorized as MRD-positive or -negative, relative to a standard PSM, when OS data are immature. Methods: Standard and response-based PSMs, estimating patient life-years (LYs) over a lifetime horizon, were developed for NDMM patients treated with bortezomib, thalidomide, and dexamethasone (BTd) with or without daratumumab as induction and consolidation therapy. In the standard PSM, LYs were determined by extrapolations from individual patient data from CASSIOPEIA. In the response-based PSM, survival was dependent on MRD status at the time of the response assessment via a landmark analysis. Cox-proportional hazard ratios from external sources and CASSIOPEIA informed the relationship for OS between MRD-positive and MRD-negative, and between patients receiving BTd and daratumumab plus BTd, respectively. Uncertainty was assessed by comparing LYs and OS extrapolations from deterministic and probabilistic analyses. Results: This response-based PSM demonstrated reduced uncertainty in long-term survival outcomes compared with the standard PSM (range across extrapolations of 3.4 and 7.7 LYs for daratumumab plus BTd and BTd, respectively, vs 14.8 and 11.8 LYs for the standard PSM). It also estimated a narrower interquartile range of LYs in the probabilistic analyses for the majority of parametric extrapolations. Discussion: Alternative methods to estimate long-term survival outcomes, such as a response-based PSM, can reduce uncertainty in modeling predictions around cost-effectiveness estimates for health technology assessment bodies and payers, thereby supporting faster market access for novel therapies with immature survival data. Conclusions: Use of MRD status in a response-based PSM reduces uncertainty in modeling long-term survival in patients with NDMM and provides a greater number of clinically plausible extrapolations compared with a standard PSM.

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter.

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.

A fungal tolerance trait and selective inhibitors proffer HMG-CoA reductase as a herbicide mode-of-action.

Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a mode of action target for the development of herbicides. Here, we present the crystal structure of a HMGR from Arabidopsis thaliana (AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enables the rational design of specific HMGR inhibitors and we develop a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.

Exploiting angled thin film vortex microfluidics for expeditious syntheses of iminosugars.

Iminosugars are important compounds in the area of carbohydrate-based therapeutics. A simple synthetic methodology utilizing the vortex fluidic thin film microfluidic reactor is effective in the synthesis of such compounds for diverse reaction types, with the optimal tilt angle of the reactor at 45° and the optimal rotational speed dependent on the nature of the liquid.

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.

Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam.

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds, as well as regulatory barriers. Of the known herbicide modes of action, 7,8-dihydropterin synthase (DHPS), which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics, and although it is still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.3 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), and we reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate that asulam and the antibiotic sulfamethoxazole have herbicidal as well as antibacterial activity, and we explore the structural basis of their potency by modeling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and indicate that pharmacokinetic or delivery differences between plants and microbes might be the best ways to safeguard this mode of action.

Vortex fluidic induced mass transfer across immiscible phases.

Mixing immiscible liquids typically requires the use of auxiliary substances including phase transfer catalysts, microgels, surfactants, complex polymers and nano-particles and/or micromixers. Centrifugally separated immiscible liquids of different densities in a 45° tilted rotating tube offer scope for avoiding their use. Micron to submicron size topological flow regimes in the thin films induce high inter-phase mass transfer depending on the nature of the two liquids. A hemispherical base tube creates a Coriolis force as a 'spinning top' (ST) topological fluid flow in the less dense liquid which penetrates the denser layer of liquid, delivering liquid from the upper layer through the lower layer to the surface of the tube with the thickness of the layers determined using neutron imaging. Similarly, double helical (DH) topological flow in the less dense liquid, arising from Faraday wave eddy currents twisted by Coriolis forces, impact through the less dense liquid onto the surface of the tube. The lateral dimensions of these topological flows have been determined using 'molecular drilling' impacting on a thin layer of polysulfone on the surface of the tube and self-assembly of nanoparticles at the interface of the two liquids. At high rotation speeds, DH flow also occurs in the denser layer, with a critical rotational speed reached resulting in rapid phase demixing of preformed emulsions of two immiscible liquids. ST flow is perturbed relative to double helical flow by changing the shape of the base of the tube while maintaining high mass transfer between phases as demonstrated by circumventing the need for phase transfer catalysts. The findings presented here have implications for overcoming mass transfer limitations at interfaces of liquids, and provide new methods for extractions and separation science, and avoiding the formation of emulsions.

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.

Inhibition of chloroplast translation as a new target for herbicides.

The rise in herbicide resistance over recent decades threatens global agriculture and food security and so discovery of new modes of action is increasingly important. Here we reveal linezolid, an oxazolidinone antibiotic that inhibits microbial translation, is also herbicidal. To validate the herbicidal mode of action of linezolid we confirmed its micromolar inhibition is specific to chloroplast translation and did not affect photosynthesis directly. To assess the herbicide potential of linezolid, testing against a range of weed and crop species found it effective pre- and post-emergence. Using structure-activity analysis we identified the critical elements for herbicidal activity, but importantly also show, using antimicrobial susceptibility assays, that separation of antibacterial and herbicidal activities was possible. Overall these results validate chloroplast translation as a viable herbicidal target.

Herbicidal activity of fluoroquinolone derivatives.

Development of herbicides with novel modes of action is crucial for weed control and to hinder herbicide resistance. An attractive novel herbicidal target is plant DNA gyrase, which has been demonstrated to be effectively inhibited by the known antimicrobial ciprofloxacin. Despite this good herbicidal activity, ciprofloxacin is not suitable as a herbicide due to its antimicrobial activity; therefore, a diverse library of analogues was analyzed to gain insight into the aspects required for herbicidal activity. This analysis revealed that significant structural modifications were tolerated and that the fluoride at C-6 and a cyclic amino group at C-7 were not crucial for herbicidal activity. The analysis also revealed that these modifications also affected the antibacterial activity with one compound demonstrating good herbicidal activity and weak antibacterial activity, against both Gram-positive and Gram-negative bacteria.

Antibiotic resistance lessons for the herbicide resistance crisis.

The challenges of resistance to antibiotics and resistance to herbicides have much in common. Antibiotic resistance became a risk in the 1950s, but a concerted global effort to manage it did not begin until after 2000. Widespread herbicide use began during the 1950s and was soon followed by an unabated rise in resistance. Here, we examine what lessons for combatting herbicide resistance could be learnt from the global, coordinated efforts of all stakeholders to avert the antibiotic resistance crisis. © 2021 Society of Chemical Industry.

Anti-Virulence Therapeutic Approaches for Neisseria gonorrhoeae.

While antimicrobial resistance (AMR) is seen in both Neisseria gonorrhoeae and Neisseria meningitidis, the former has become resistant to commonly available over-the-counter antibiotic treatments. It is imperative then to develop new therapies that combat current AMR isolates whilst also circumventing the pathways leading to the development of AMR. This review highlights the growing research interest in developing anti-virulence therapies (AVTs) which are directed towards inhibiting virulence factors to prevent infection. By targeting virulence factors that are not essential for gonococcal survival, it is hypothesized that this will impart a smaller selective pressure for the emergence of resistance in the pathogen and in the microbiome, thus avoiding AMR development to the anti-infective. This review summates the current basis of numerous anti-virulence strategies being explored for N. gonorrhoeae.

Sub-micron moulding topological mass transport regimes in angled vortex fluidic flow.

Shear stress in dynamic thin films, as in vortex fluidics, can be harnessed for generating non-equilibrium conditions, but the nature of the fluid flow is not understood. A rapidly rotating inclined tube in the vortex fluidic device (VFD) imparts shear stress (mechanical energy) into a thin film of liquid, depending on the physical characteristics of the liquid and rotational speed, ω, tilt angle, θ, and diameter of the tube. Through understanding that the fluid exhibits resonance behaviours from the confining boundaries of the glass surface and the meniscus that determines the liquid film thickness, we have established specific topological mass transport regimes. These topologies have been established through materials processing, as spinning top flow normal to the surface of the tube, double-helical flow across the thin film, and spicular flow, a transitional region where both effects contribute. The manifestation of mass transport patterns within the film have been observed by monitoring the mixing time, temperature profile, and film thickness against increasing rotational speed, ω. In addition, these flow patterns have unique signatures that enable the morphology of nanomaterials processed in the VFD to be predicted, for example in reversible scrolling and crumbling graphene oxide sheets. Shear-stress induced recrystallisation, crystallisation and polymerisation, at different rotational speeds, provide moulds of high-shear topologies, as 'positive' and 'negative' spicular flow behaviour. 'Molecular drilling' of holes in a thin film of polysulfone demonstrate spatial arrangement of double-helices. The grand sum of the different behavioural regimes is a general fluid flow model that accounts for all processing in the VFD at an optimal tilt angle of 45°, and provides a new concept in the fabrication of novel nanomaterials and controlling the organisation of matter.

Improved herbicide discovery using physico-chemical rules refined by antimalarial library screening.

Herbicides have physico-chemical properties not unlike orally-delivered human drugs, but are known to diverge in their limits for proton donors, partition coefficients and molecular weight. To further refine rules specific for herbicides, we exploited the close evolutionary relationship between Plasmodium falciparum and plants by screening the entire Malaria Box, a chemical library of novel chemical scaffolds with activity against the blood stage of P. falciparum. Initial screening against Arabidopsis thaliana on agar media and subsequently on soil demonstrated the crucial nature of log P and formal charge are to active molecules. Using this information, a weighted scoring system was applied to a large chemical library of liver-stage effective antimalarial leads, and of the six top-scoring compounds, one had potency comparable to that of commercial herbicides. This novel compound, MMV1206386, has no close structural analogues among commercial herbicides. Physiological profiling suggested that MMV1206386 has a new mode of action and overall demonstrates how weighted rules can help during herbicide discovery programs.

Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi.

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.