Structure and antigenicity of divergent Henipavirus fusion glycoproteins.

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiang virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Recent insights into mechanisms of cellular toxicity and cell recognition associated with the ABC family of pore-forming toxins.

ABC toxins are pore-forming toxins characterised by the presence of three distinct components assembled into a hetero-oligomeric toxin complex ranging in size from 1.5-2.5 MDa. Most ABC toxins studied to date appear to be insecticidal toxins, although genes predicted to encode for homologous assemblies have also been found in human pathogens. In insects, they are delivered to the midgut either directly via the gastrointestinal tract, or via a nematode symbiont, where they attack the epithelial cells and rapidly trigger widespread cell death. At the molecular level, the homopentameric A subunit is responsible for binding to lipid bilayer membranes and introducing a protein translocation pore, through which a cytotoxic effector - encoded at the C-terminus of the C subunit - is delivered. The B subunit forms a protective cocoon that encapsulates the cytotoxic effector, part of which is contributed by the N-terminus of the C subunit. The latter also includes a protease motif that cleaves the cytotoxic effector, releasing it into the pore lumen. Here, we discuss and review recent studies that begin to explain how ABC toxins selectively target specific cells, establishing host tropism, and how different cytotoxic effectors trigger cell death. These findings allow for a more complete understanding of how ABC toxins function in an in vivo context, which in turn provides a stronger foundation for understanding how they cause disease in invertebrate (and potentially also vertebrate) hosts, and how they might be re-engineered for therapeutic or biotechnological purposes.

Genome-Guided Analysis of Seven Weed Species Reveals Conserved Sequence and Structural Features of Key Gene Targets for Herbicide Development.

Herbicides are commonly deployed as the front-line treatment to control infestations of weeds in native ecosystems and among crop plants in agriculture. However, the prevalence of herbicide resistance in many species is a major global challenge. The specificity and effectiveness of herbicides acting on diverse weed species are tightly linked to targeted proteins. The conservation and variance at these sites among different weed species remain largely unexplored. Using novel genome data in a genome-guided approach, 12 common herbicide-target genes and their coded proteins were identified from seven species of Weeds of National Significance in Australia: Alternanthera philoxeroides (alligator weed), Lycium ferocissimum (African boxthorn), Senecio madagascariensis (fireweed), Lantana camara (lantana), Parthenium hysterophorus (parthenium), Cryptostegia grandiflora (rubber vine), and Eichhornia crassipes (water hyacinth). Gene and protein sequences targeted by the acetolactate synthase (ALS) inhibitors and glyphosate were recovered. Compared to structurally resolved homologous proteins as reference, high sequence conservation was observed at the herbicide-target sites in the ALS (target for ALS inhibitors), and in 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (target for glyphosate). Although the sequences are largely conserved in the seven phylogenetically diverse species, mutations observed in the ALS proteins of fireweed and parthenium suggest resistance of these weeds to ALS-inhibiting and other herbicides. These protein sites remain as attractive targets for the development of novel inhibitors and herbicides. This notion is reinforced by the results from the phylogenetic analysis of the 12 proteins, which reveal a largely consistent vertical inheritance in their evolutionary histories. These results demonstrate the utility of high-throughput genome sequencing to rapidly identify and characterize gene targets by computational methods, bypassing the experimental characterization of individual genes. Data generated from this study provide a useful reference for future investigations in herbicide discovery and development.

Structural basis of resistance to herbicides that target acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS) is the target for more than 50 commercial herbicides; first applied to crops in the 1980s. Since then, 197 site-of-action resistance isolates have been identified in weeds, with mutations at P197 and W574 the most prevalent. Consequently, AHAS is at risk of not being a useful target for crop protection. To develop new herbicides, a functional understanding to explain the effect these mutations have on activity is required. Here, we show that these mutations can have two effects (i) to reduce binding affinity of the herbicides and (ii) to abolish time-dependent accumulative inhibition, critical to the exceptional effectiveness of this class of herbicide. In the two mutants, conformational changes occur resulting in a loss of accumulative inhibition by most herbicides. However, bispyribac, a bulky herbicide is able to counteract the detrimental effects of these mutations, explaining why no site-of-action resistance has yet been reported for this herbicide.

Herbicides That Target Acetohydroxyacid Synthase Are Potent Inhibitors of the Growth of Drug-Resistant Candida auris.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, is the target for more than 50 commercially available herbicides, and is a promising target for antimicrobial drug discovery. Herein, we have expressed and purified AHAS from Candida auris, a newly identified human invasive fungal pathogen. Thirteen AHAS inhibiting herbicides have Ki values of <2 µM for this enzyme, with the most potent having Ki values of <32 nM. Six of these compounds exhibited MIC50 values of <1 µM against C. auris (CBS10913 strain) grown in culture, with bensulfuron methyl (BSM) being fungicidal and the most potent (MIC50 of 0.090 µM) in defined minimal media. The MIC50 value increases to 0.90 µM in media enriched by the addition of branched-chain amino acids at the expected concentration in the blood serum. The sessile MIC50 for BSM is 0.6 µM. Thus, it is also an excellent inhibitor of the growth of C. auris biofilms. BSM is nontoxic in HEK-293 cells at concentrations >100 µM and thus possesses a therapeutic index of >100. These data suggest that targeting AHAS is a viable strategy for treating C. auris infections.

Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections.

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.