Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. In addition, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium. The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa-infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for the development of effective alternatives to traditional antibiotics in the fight against this resilient pathogen.

The Gram-positive pathogen Streptococcus pneumoniae, like the majority of bacteria, contains a peptidoglycan-based cell wall whose structure is highly dependent on the action of penicillin-binding proteins (PBPs). While the β-lactam antibiotics have been employed as an antimicrobial strategy for nearly a century, much remains unclear about how inhibitor structure informs potency and PBP isoform selectivity. Here, we obtained high-resolution structures (<2Å) of S. pneumoniae PBP1b cocrystallized with 6 β-lactams. Surprisingly, 2 structures feature a noncanonical conformation of the covalent "acyl-enzyme complex." To clarify how protein-ligand interactions mediate inhibitor binding, we applied molecular modeling and molecular mechanics-based dynamics analyses. Our analyses illustrate how seemingly minimal changes to inhibitor structure modulate β-lactam binding mode and inhibitor potency, as described by the metric kinact/KI. Furthermore, we demonstrate that persistent interaction in the covalent acyl-enzyme complex between the inhibitor carboxylate and a highly conserved three-residue motif is not fully predictive of kinact/KI for PBP1b. In silico modeling suggests that the noncovalent preacyl complex may leverage this interaction, but a postacylation change in ligand conformation may accompany acylation in some inhibitors. The elucidation of key PBP1b ligand-receptor interactions pre- and postacylation will inform the rational design of novel PBP inhibitors and probes.

Abstract Alix is a key adaptor protein of the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery. Although Alix-mediated recruitment of the ESCRT-III subunit CHMP4 is well established, the molecular mechanisms underlying Alix activation and ESCRT-III polymerization remain poorly understood. Here, we present the crystal structure of the dimeric Alix V-domain (Alix-V) in complex with a llama nanobody. Dimerization is mediated by domain swapping, generating an X-shaped flexible conformation. We demonstrate that Alix forms dimers in vivo and provide evidence for the recruitment of dimeric Alix to plasma membrane repair sites and the cytokinetic midbody. Notably, a mutation disrupting Alix dimerization impairs plasma membrane repair. Furthermore, high-speed AFM experiments reveal that dimeric Alix, but not the monomeric form, nucleates CHMP4B filament polymerization. Our data establish that dimeric Alix is the active form responsible for nucleating two ESCRT-III CHMP4 filaments, whose geometry creates a platform for the recruitment of downstream ESCRT-III components required to assemble the active membrane remodeling and fission machinery.

Monobactams, a subclass of β-lactam antibiotics with a monocyclic scaffold, are uniquely resistant to hydrolysis by metallo-β-lactamases, providing a distinct therapeutic advantage. Here, we report an in silico-based structure-activity relationship (SAR) investigation of aztreonam-related monobactams. A focused library of monobactam derivatives was synthesized and evaluated for inhibition of penicillin-binding proteins (PBPs) and antibacterial activity. Ten compounds, including aztreonam, were crystallized with truncated PBP1b from Streptococcus pneumoniae, used as a model PBP. Potent PBP1b inhibitors were developed, although high enzymatic potency was not always reflected in strong antibacterial activity. Certain derivatives showed activity against Staphylococcus aureus, which is typically resistant to monobactams. 2D similarity search identified potent inhibitors active against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Crystal structures revealed previously unrecognized binding interactions, including a halogen bond with a conserved threonine residue, underscoring the potential of these interactions to support the development of more potent PBP inhibitors.

Acinetobacter baumannii is a major human pathogen responsible for a growing number of multi-antibiotic-resistant infections, and of critical priority for the World Health Organization (WHO). A. baumannii employs a type II secretion system (T2SS) to secrete toxins extracellularly to enable cytotoxicity and colonization. Lipase LipA, secreted by the A. baumannii T2SS, is required for virulence and fitness, and in the periplasm is maintained in an active state by its essential foldase, LipB. Here we report that LipA is able to recognize lipids of different chain lengths at extremes of pH and temperature, thanks to its stabilization by LipB through an extended, highly helical “embrace.” A vast bioinformatic analysis indicates that LipB-like foldases are widespread over numerous proteobacteria, and thus the extended foldase architecture shown here could be widespread. These results provide new insight into A. baumannii’s adaptability as a pathogen in different environments and could facilitate the development of novel antibacterials.

Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting to isolate, from cystic fibrosis patients, human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. Further, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium. The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV's scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa-infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for developing effective alternatives to traditional antibiotics in the fight against this resilient pathogen. ### Competing Interest Statement The authors have declared no competing interest.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of β-lactams by β-lactamases, (ii) the interaction of drugs belonging to the β-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

In the final phases of bacterial cell wall synthesis, penicillin-binding proteins (PBPs) catalyze the cross-linking of peptidoglycan. For many decades, effective and non-toxic β-lactam antibiotics have been successfully used as mimetics of the d-Ala-d-Ala moiety of the natural substrate and employed as irreversible inhibitors of PBPs. In the years following their discovery, the emergence of resistant bacteria led to a decline in their clinical efficacy. Using Staudinger cycloaddition, we synthesized a focused library of novel monocyclic β-lactams in which different substituents were introduced at the C4 position of the β-lactam ring, at the C3 amino position, and at the N1 lactam nitrogen. In biochemical assays, the compounds were evaluated for their inhibitory effect on the model enzyme PBP1b from Streptococcus pneumoniae. Upon investigation of the antibacterial activity of the newly prepared compounds against ESKAPE pathogens, some compounds showed moderate inhibition. We also examined their reactivity and selectivity in a biochemical assay with other enzymes that have a catalytic serine in the active site, such as human cholinesterases, where they also showed no inhibitory activity, highlighting their specificity for bacterial targets. These compounds form the basis for further work on new monocyclic β-lactams with improved antibacterial activity.

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaen-zymes to generate diverse metabolites with many applications.

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.

Bacterial cell wall formation is essential for cellular survival and morphogenesis. The peptidoglycan (PG), a heteropolymer that surrounds the bacterial membrane, is a key component of the cell wall, and its multistep biosynthetic process is an attractive antibacterial development target. Penicillin-binding proteins (PBPs) are responsible for cross-linking PG stem peptides, and their central role in bacterial cell wall synthesis has made them the target of successful antibiotics, including β-lactams, that have been used worldwide for decades. Following the discovery of penicillin, several other compounds with antibiotic activity have been discovered and, since then, have saved millions of lives. However, since pathogens inevitably become resistant to antibiotics, the search for new active compounds is continuous. The present review highlights the ongoing development of inhibitors acting mainly in the transpeptidase domain of PBPs with potential therapeutic applications for the development of new antibiotic agents. Both the critical aspects of the strategy, design, and structure–activity relationships (SAR) are discussed, covering the main published articles over the last 10 years. Some of the molecules described display activities against main bacterial pathogens and could open avenues toward the development of new, efficient antibacterial drugs.

β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four β-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the β-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

Bacteria of the genus Psychrobacter are known for their psychrophilic characteristics, being extremophilic organisms capable of surviving and reproducing in hostile environments of low temperature and high pressure. Among many of the genus characteristics, there is the ability to produce enzymes and molecules of industrial biotechnology importance, such as pigments and proteins related to heavy metal bioremediation. The bacterium strain Psychrobacter nivimaris LAMA 639 was isolated from sediments from the Walvis Ridge ocean crest at a depth of 4.400 m (33.40 S 2.35 E). It is a nonmotile, halotolerant, cream-colored gram-negative aerobic bacterium. Its cultivation was performed in marine agar plates and inoculated into test tubes with NaCl at an optimal temperature of 30 °C and with shaking at 100 rpm. Genome extraction was performed with the DNeasy Blood & Tissue Kit (QIAGEN®). Sequencing was performed by Macrogen using the NovaSeq® 6000 platform (Illumina) applying the whole genome shotgun (WGS) method. Thereafter, 14.712.526 reads of 151 bp were generated, totaling 2.2 G bp with a GC content of 42.9%. Assembly and mapping were performed with a CLC Genomics Workbench. The best assembly considered was the one with the lowest number of contigs and the highest base length pair. The assemblies were evaluated using QUAST, and the best resulting variant was selected for annotation. Genome annotation was performed with RAST and PATRIC; the antiSMASH tool was used for secondary metabolites; NaPDoS was used for domains; and three-dimensional structural prediction of relevant proteins was performed using Phyre2. Annotation with ClassicRAST generated 2,891 coding sequences (CDSs) distributed in 402 subsystems. Annotation with PATRIC generated 2,896 coding sequences, among them 776 hypothetical proteins. The antiSMASH tool visualized a beta-lactone cluster in contig 06. In the search for natural products with NaPDoS, two ketosynthase domains were identified. The search for relevant proteins was performed using the AMFEP list as a criterion. From these data, 34 possible enzymes with biotechnological potential were found. Finally, the organism is presented as a new reference regarding the potential of deep-sea marine bacteria, demonstrating that, from the annotated and cured genome, it is possible to find in its genetic repertory products of interest for biotechnological applications.

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC’s ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.