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For centuries, Gram-negative pathogens have infected the human population and been responsible for numerous diseases in animals and plants. Despite advancements in therapeutics, Gram-negative pathogens continue to evolve, with some having developed multi-drug resistant phenotypes. For the successful control of infections caused by these bacteria, we need to widen our understanding of the mechanisms of host-pathogen interactions. Gram-negative pathogens utilise an array of effector proteins to hijack the host system to survive within the host environment. These proteins are secreted into the host system via various secretion systems, including the integral Type III secretion system (T3SS). The T3SS spans two bacterial membranes and one host membrane to deliver effector proteins (virulence factors) into the host cell. This multifaceted process has multiple layers of regulation and various checkpoints. In this review, we highlight the multiple strategies adopted by these pathogens to regulate or maintain virulence via the T3SS, encompassing the regulation of small molecules to sense and communicate with the host system, as well as master regulators, gatekeepers, chaperones, and other effectors that recognise successful host contact. Further, we discuss the regulatory links between the T3SS and other systems, like flagella and metabolic pathways including the tricarboxylic acid (TCA) cycle, anaerobic metabolism, and stringent cell response., (© 2024 Cambridge Philosophical Society.)

The accumulation of tau fibrils is associated with neurodegenerative diseases, which are collectively termed tauopathies. Cryo-EM studies have shown that the packed fibril core of tau adopts distinct structures in different tauopathies, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. A subset of tauopathies are linked to missense mutations in the tau protein, but it is not clear whether these mutations impact the structure of tau fibrils. To answer this question, we developed a high-throughput protein purification platform and purified a panel of 37 tau variants using the full-length 0N4R splice isoform. Each of these variants was used to create fibrils in vitro , and their relative structures were studied using a high-throughput protease sensitivity platform. We find that a subset of the disease-associated mutations form fibrils that resemble wild-type tau, while others are strikingly different. The impact of mutations on tau structure was not clearly associated with either the location of the mutation or the relative kinetics of fibril assembly, suggesting that tau mutations alter the packed core structures through a complex molecular mechanism. Together, these studies show that single-point mutations can impact the assembly of tau into fibrils, providing insight into its association with pathology and disease.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and is involved in iron oxidation, storage, and mineralization. Although several structures of human ferritins and bacterioferritins have been solved, there is still no complete structure that shows both the trapped Fe-biomineral cluster and the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, structural details on the biomineralization process (i.e. the formation of the mineral itself) are generally lacking. Here, we report the cryo-electron microscopy (cryo-EM) structures of apoform and biomineral bound form (holoforms) of the Streptomyces coelicolor bacterioferritin (ScBfr) nanocage and the subunit crystal structure. The holoforms show different stages of Fe-biomineral accumulation inside the nanocage, in which the connections exist in two of the fourfold channels of the nanocage between the C-terminal of the ScBfr monomers and the Fe-biomineral cluster. The mutation and truncation of the bacterioferritin residues involved in these connections significantly reduced the iron and phosphate binding in comparison with those of the wild type and together explain the underlying mechanism. Collectively, our results represent a prototype for the bacterioferritin nanocage, which reveals insight into its biomineralization and the potential channel for bacterioferritin-associated iron trafficking., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

This study reports the isolation of a new Chromobacterium haemolyticum strain named WI5 from a hydroponic farming facility. WI5 exhibited remarkable bacterial antagonistic properties, eliminating Salmonella, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus (initial inoculum load ∼10 5  CFU/ml) in dual-species co-culture biofilms. Antagonism was strictly contact-dependent and highly influenced by nutrient availability. Next, we identified a complete suite of putative Type VI secretion system (T6SS) genes in the WI5 genome, annotated the gene locus architecture, and determined the crystal structure of hallmark T6SS tube protein Hcp1, which revealed a hexameric ring structure with an outer and inner diameter of 77 and 45 Å, respectively. Structural comparison with homologs showed differences in the key loops connecting the β-strands in which the conserved residues are located, suggesting a role of these residues in the protein function. The T6SS is well-known to facilitate interbacterial competition, and the putative T6SS characterized herein might be responsible for the remarkable antagonism by C. haemolyticum WI5. Collectively, these findings shed light on the nature of bacterial antagonism and a putative key virulence determinant of C. haemolyticum, which might aid in further understanding its potential ecological role in natural habitats., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Gram-negative pathogens like Enteropathogenic Escherichia coli (EPEC) utilize the type three secretion system (T3SS) to translocate various effector proteins that are needed to "hijack" the host system for pathogenic survival. Specialized T3SS chaperones inside bacterial cells stabilize these effector proteins and facilitate their translocation. CesT is a unique multi-cargo chaperone that interacts with and translocates ~10 different effector proteins. Here, we report the specific interaction between CesT and its key effector, NleH2, and explore the potential role of NleH2 as a kinase for CesT phosphorylation. First, we identified the chaperone-binding domain (CBD; 19-97aa) of NleH2, and mapped the specific interaction sites for both CesT and NleH2. The N- and C-terminal residues of the CBD interact with the dimeric interface of CesT. Further, we compared the CesT binding to NleH2, to that of another key effector Tir and with the global carbon regulator CsrA. Notably, the effectors have the binding regions at the β-sheet core and dimer interface of CesT, whereas the CsrA regulator interacts predominantly through the C-terminal region, which is found ~17 Å away from the effectors-binding sites. Next, we showed that NleH2 remains an active kinase even as a complex with CesT and is responsible for its autophosphorylation as well as phosphorylation of CesT at Tyr153. Collectively, our findings enhance the understanding of the role of multi-cargo chaperone CesT in orchestrating effector translocation through T3SS., (© 2021 The Protein Society.)