Biology subjects

Roper, D. I.

Publications and source records attributed to Roper, D. I..

6 recordsLinked to original sources

bioRxiv2025

Regulation of the essential peptidoglycan hydrolytic complex FtsEX-PcsB during Streptococcus pneumoniae cell division

In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae, the essential cell-division-associated peptidoglycan (PG) hydrolase PcsB interacts directly with the membrane-bound FtsEX complex (1-3). PcsB contains a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain responsible for PG hydrolysis, and a coiled-coil domain required for interaction with FtsEX (1,4). ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane, which ultimately regulates the PG hydrolase on the outside of the cell (5). In this work we show that the CHAP domain of PcsB predominately functions as an D-iso-Glutaminyl-Lysyl D,L-endopeptidase, with substrate specificity for Lys-containing, amidated PG. The catalytic activity of PcsB is regulated by conformational changes of the coiled-coil region and by a short helical region immediately adjacent to the CHAP domain, to guard against PcsB hydrolytic activation outside of its cell division specific functional requirement. This work supports a model for the overall biological activity of the FtsEX-PcsB complex, allowing stem peptide cleavage that splits the septal disk and marks a region of the peptidoglycan sacculus for subsequent cell division remodelling.

biochemistry↗

bioRxiv2024

Delivery determinants of an Acinetobacter baumannii type VI secretion system bifunctional peptidoglycan hydrolase.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is a common cause of nosocomial infections. The increasing development of antibiotic resistance in this organism is a global health concern. The A. baumannii clinical isolate AB307-0294 produces a type VI secretion system (T6SS) that delivers three antibacterial cargo effector proteins (Tse15, Tde16 and Tae17) that give this strain a competitive advantage against other bacteria in polymicrobial environments. These effectors are delivered via specific non-covalent interactions with the T6SS needle tip proteins VgrG15, VgrG16 and VgrG17, respectively. Here we determine the molecular function of the Tae17 effector protein and define the regions of Tae17 and VgrG17 essential for its delivery. Specifically, we show that Tae17 is a multidomain, bifunctional peptidoglycan-degrading enzyme. Tae17 has both lytic transglycosylase activity, which targets the peptidoglycan sugar backbone, and amidase activity, which targets the sugar-peptide bonds. Moreover, we show that the transglycosylase activity was more important for killing Escherichia coli. Using deletion constructs and bacterial two-hybrid analyses, we identify that amino acids 1051-1085 of the VgrG17 needle tip protein and amino acids 1-162 of the Tae17 effector protein are necessary for the Tae17:VgrG17 interaction. Furthermore, we identify the VgrG17 amino acids G1069 and W1075 as crucial for the delivery of Tae17; the first time such specific delivery determinants of T6SS cargo effectors have been defined. This study provides molecular insight into how the T6SS allows A. baumannii strains to gain dominance in polymicrobial communities and thus improve their chances of survival and transmission. IMPORTANCEWe have shown that the Acinetobacter baumannii T6SS effector Tae17 is a modular, bifunctional, peptidoglycan-degrading enzyme that has both lytic transglycosylase and amidase activity. Both activities contribute to the ability to degrade peptidoglycan, but the glycosyltransferase activity was more important for the interbacterial killing of Escherichia coli. We have defined the specific regions of Tae17 and its cognate delivery protein VgrG17 that are necessary for the non-covalent interactions and, for the first time, identified specific amino acids essential for delivery. This work contributes to our molecular understanding of bacterial competition strategies in polymicrobial environments and may provide a window to the design of new therapeutic approaches for combating infection by A. baumannii.

microbiology↗

bioRxiv2024

Discovery and chemical optimisation of a Potent, Bi-cyclic (Bicycle(R)) Antimicrobial Inhibitor of Escherichia coli PBP3

Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of {beta}-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of novel, bicyclic peptide (Bicycle(R)) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent a novel non-{beta}-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors.

biochemistry↗

bioRxiv2024

Structural insights into peptidoglycan hydrolysis by the FtsEX system in Escherichia coli during cell division

Bacterial cell division relies on precise peptidoglycan (PG) remodelling, a process orchestrated by the FtsEX complex. Comprised of FtsE and FtsX, this complex collaborates with EnvC, a periplasmic lytic enzyme activator, to regulate septal PG hydrolysis by amidases like AmiB. While recent structural investigations, particularly of Pseudomonas aeruginosa FtsEX (PaeFtsEX), have shed light on complex interactions and proposed activation mechanisms, the structural intricacies governing PG degradation by the FtsEX complex and EnvC in Escherichia coli cytokinesis remain unexplored. In this study, we present a comprehensive biochemical and structural analysis of E. coli FtsEX complexes, unveiling a key role for ATP in complex stabilization that extends across bacterial species. Upon EnvC binding, ATPase activity markedly increases. High-resolution structures of EcoFtsEX, both in the presence and absence of EnvC, reveal a symmetrical conformation of EcoFtsEX capable of accommodating the inherent asymmetry of EnvC, mediated by flexible loops within the periplasmic domain. Our negative-staining imaging showcases an elongated EcoFtsEX/EnvC/AmiB complex reminiscent of the PaeFtsEX system. These findings collectively provide intricate insights into the regulation of PG cleavage by FtsEX in E. coli - a pivotal model system used in pilot genetic studies, suggesting a conserved mechanism for precise hydrolase activation in bacteria.

microbiology↗

bioRxiv2022

Plant peptidoglycan precursor biosynthesis: Conservation between moss chloroplasts and Gram negative bacteria

An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram positive bacteria. To establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein, UDP-MurNAc-tripeptide ligase (MurE), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.

evolutionary biology↗

bioRxiv2019

Structure-guided enhancement of selectivity of chemical probe inhibitors targeting bacterial seryl-tRNA synthetase

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than two orders of magnitude compared to their human homologue, demonstrating a route to selective chemical inhibition of these bacterial targets.

biochemistry↗