The global increase in multi-drug-resistant bacteria is severely impacting our ability to effectively treat common infections. For Gram-negative bacteria, their intrinsic and acquired resistance mechanisms are heightened by their unique cell wall structure. The cell wall, while being a target of some antibiotics, represents a barrier due to the inability of most antibacterial compounds to traverse and reach their intended target. This means that its composition and resulting mechanisms of resistance must be considered when developing new therapies. Here, we discuss potential antibiotic targets within the most well-characterised resistance mechanisms associated with the cell wall in Gram-negative bacteria, including the outer membrane structure, porins and efflux pumps. We also provide a timely update on the current progress of inhibitor development in these areas. Such compounds could represent new avenues for drug discovery as well as adjuvant therapy to help us overcome antibiotic resistance.

Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double‐stranded DNA virus belonging to the poxviridae that causes mild monkeypox‐like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl‐2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl‐2 fold comprising 7 α‐helices and utilizes the canonical Bcl‐2 binding groove to engage proapoptotic host cell Bcl‐2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain‐swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain‐swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus‐mediated inhibition of host cell apoptosis and reveal the capacity of Bcl‐2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. Database Structural data are available in the Protein Data Bank (PDB) under the accession numbers http://6TPQ, http://6TQQ and http://6TRR.

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. Databases PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6P90.

The rise of antibiotic resistance combined with the lack of new products entering the market has led to bacterial infections becoming one of the biggest threats to global health. Therefore, there is an urgent need to identify novel antibiotic targets, such as dihydrodipicolinate synthase (DHDPS), an enzyme involved in the production of essential metabolites in cell wall and protein synthesis. Here, we utilised a 7‐residue sequence motif to identify mis‐annotation of multiple DHDPS genes in the high‐priority Gram‐negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. We subsequently confirmed these mis‐annotations using a combination of enzyme kinetics and X‐ray crystallography. Thus, this study highlights the need to ensure genes encoding promising drug targets, like DHDPS, are annotated correctly, especially for clinically important pathogens.PDB ID6UE0.

22Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular 23 organisms to counter microbial threats. Tanapoxvirus (TPV) is a large double-stranded DNA 24 virus belonging to the poxviridae that causes mild Monkeypox-like infections in humans and 25 primates. TPV encodes for a putative apoptosis inhibitory protein 16L. We now show that 26 TPV16L is able to bind to a range of peptides spanning the BH3 motif of human pro-27 apoptotic Bcl-2 proteins, and is able to counter growth arrest of yeast induced by human Bak 28 and Bax. We then determined the crystal structures of TPV16L bound to three identified 29 interactors, Bax, Bim and Puma BH3. TPV16L adopts a globular Bcl-2 fold comprising 7 α-30 helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-31 2 proteins. Unexpectedly, TPV16L is able to adopt both a monomeric as well as a domain-32 swapped dimeric topology where the α1 helix from one protomer is swapped into a 33 neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand 34 binding groove in TPV16L remains unchanged from monomer to domain-swapped dimer. 35Our results provide a structural and mechanistic basis for tanapoxvirus mediated inhibition of 36 host cell apoptosis, and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric 37 states whilst maintaining the canonical ligand binding groove in an unchanged state. 38 39 40