Chemoproteomics validates selective targeting of M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant (A-M1) and (A-M1) M1 metalloaminopeptidases, with selectivity over other and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant ( A-M1) and ( A-M1) M1 metalloaminopeptidases, with selectivity over other and human aminopeptidases, and displayed excellent antimalarial activity with no significant host cytotoxicity.

Dissecting EXP2 sequence requirements for protein export in malaria parasites.

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in EXP2, however, has an additional function in parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'β-signal' imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis.

Design, Synthesis, and Evaluation of Cephamycin-Based Antisporulation Agents targeting .

With the aim of discovering small molecule inhibitors of the sporulation process in , we prepared a series of C-7 α-(4-substituted-1-1,2,3-triazol-1-yl)acetamide analogues of cefotetan, a known inhibitor of the sporulation-specific protein target SpoVD. These analogues were evaluated using both binding assays with SpoVD and antisporulation assays against . Further design concepts were aided utilizing the predicted docking scores (DS) using both AlphaFold (AF) models, and a crystal structure of the SpoVD protein (PDB 7RCZ).

Understanding the structure and function of Plasmodium aminopeptidases to facilitate drug discovery.

Malaria continues to be the most widespread parasitic disease affecting humans globally. As parasites develop drug resistance at an alarming pace, it has become crucial to identify novel drug targets. Over the last decade, the metalloaminopeptidases have gained importance as potential targets for new antimalarials.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of . A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes.

Structure-based development of potent Plasmodium falciparum M1 and M17 aminopeptidase selective and dual inhibitors via S1'-region optimisation.

Malaria remains a global health threat and growing resistance to artemisinin-based therapies calls for therapeutic agents with novel mechanisms of action. The Plasmodium spp M1 and M17 metalloaminopeptidases have been identified as attractive new antimalarial drug targets as inhibition of these enzymes results in antiplasmodial activity. Previously identified novel hydroxamic acid 2 as a moderate inhibitor of PfA-M1 and PfA-M17 and a potent inhibitor of P.

Genetic and chemical validation of aminopeptidase A-M17 as a drug target in the hemoglobin digestion pathway.

the causative agent of malaria, remains a global health threat as parasites continue to develop resistance to antimalarial drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. parasites infect human red blood cells where they digest the host's main protein constituent, hemoglobin.

A metal ion-dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase.

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined.

The Carbapenemase BKC-1 from Klebsiella pneumoniae Is Adapted for Translocation by Both the Tat and Sec Translocons.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. β-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of β-lactam drugs is to populate the periplasmic space with β-lactamases.

Mapping the substrate specificity of the Plasmodium M1 and M17 aminopeptidases.

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei.

X-ray crystal structure and specificity of the Toxoplasma gondii ME49 TgAPN2.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii that currently has few therapeutic options. The M1 aminopeptidase enzymes have been shown to be attractive targets for anti-parasitic agents and/or vaccine candidates, suggesting potential to re-purpose inhibitors between parasite M1 aminopeptidase targets. The M1 aminopeptidase TgAPN2 has been suggested to be a potential new drug target for toxoplasmosis.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E.

Reductive evolution in outer membrane protein biogenesis has not compromised cell surface complexity in Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterial pathogen that chronically inhabits the human stomach. To survive and maintain advantage, it has evolved unique host-pathogen interactions mediated by Helicobacter-specific proteins in the bacterial outer membrane. These outer membrane proteins (OMPs) are anchored to the cell surface via a C-terminal β-barrel domain, which requires their assembly by the β-barrel assembly machinery (BAM).

Structural basis for substrate selection by the translocation and assembly module of the β-barrel assembly machinery.

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the β-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex.

Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria.

Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology.

Identification of BamC on the Surface of E. coli.

In order to relate the structural architecture of the BAM complex to its function in outer membrane protein assembly, the arrangement of each component within the complex is vital. This chapter explores the structure and topology of BamC, using a range of biochemical techniques to probe the topology and surface exposure.

Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes.

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR).

Transcriptional activation of the mrkA promoter of the Klebsiella pneumoniae type 3 fimbrial operon by the c-di-GMP-dependent MrkH protein.

The Gram-negative bacterial pathogen Klebsiella pneumoniae forms biofilms to facilitate colonization of biotic and abiotic surfaces. The formation of biofilms by K. pneumoniae requires the expression of type 3 fimbriae: elongate proteinaceous filaments extruded by a chaperone-usher system in the bacterial outer membrane.

Assembly of β-barrel proteins into bacterial outer membranes.

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo.

Evolution of the β-barrel assembly machinery.

Proteins from the Omp85 family have roles in membrane biogenesis, and the archetypal protein of this family is the bacterial outer membrane protein BamA. Through evolution, BamA has acquired membrane protein partner subunits, but distinct partner subunits are evident in the various bacterial lineages. As a result, experimental work on several species of bacteria has revealed varietal forms of the β-barrel assembly machinery (BAM complex).

A bioinformatic strategy for the detection, classification and analysis of bacterial autotransporters.

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion.