Bacterial gasdermins reveal an ancient mechanism of cell death

Ancient origin of cell death Gasdermins are cell death proteins in mammals that form membrane pores in response to pathogen infection. Johnson et al . report that diverse bacteria encode structural and functional homologs of mammalian gasdermins. Like their mammalian counterparts, bacterial gasdermins are activated by caspase-like proteases, oligomerize into large membrane pores, and defend against pathogen—in this case, bacteriophage—infection. Proteolytic activation occurs through the release of a short inhibitory peptide, and many bacterial gasdermins are lipidated to facilitate membrane pore formation. Pyroptotic cell death, a central component of mammalian innate immunity, thus has a shared origin with an ancient antibacteriophage defense system. —SMH

Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures

To dissect the antibiotic role of nanostructures from chemical moieties belligerent to both bacterial and mammalian cells, here we show the antimicrobial activity and cytotoxicity of nanoparticle-pinched polymer brushes (NPPBs) consisting of chemically inert silica nanospheres of systematically varied diameters covalently grafted with hydrophilic polymer brushes that are non-toxic and non-bactericidal. Assembly of the hydrophilic polymers into nanostructured NPPBs doesn’t alter their amicability with mammalian cells, but it incurs a transformation of their antimicrobial potential against bacteria, including clinical multidrug-resistant strains, that depends critically on the nanoparticle sizes. The acquired antimicrobial potency intensifies with small nanoparticles but subsides quickly with large ones. We identify a threshold size (dsilica ~ 50 nm) only beneath which NPPBs remodel bacteria-mimicking membrane into 2D columnar phase, the epitome of membrane pore formation. This study illuminates nanoengineering as a viable approach to develop nanoantibiotics that kill bacteria upon contact yet remain nontoxic when engulfed by mammalian cells.

Membrane interactions and uncoating of Aichi virus, a picornavirus that lacks a VP4

Kobuviruses are an unusual and poorly characterised genus within the picornavirus family, and can cause gastrointestinal enteric disease in humans, livestock and pets. The human Kobuvirus, Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of five years, however this is a very rare occurrence. During the assembly of most picornaviruses (e.g. poliovirus, rhinovirus and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, Kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure and enhances permeabilisation of model membranes. Furthermore, using peptides we demonstrate that the N-terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. Importance: To initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small non-enveloped RNA viruses includes significant human and animal pathogens and are also models to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses re-arrange their capsids and induce membrane permeability in the absence of VP4. Here we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

A Physics-Informed Convolutional Neural Network with Custom Loss Functions for Porosity Prediction in Laser Metal Deposition

Physics-informed machine learning is emerging through vast methodologies and in various applications. This paper discovers physics-based custom loss functions as an implementable solution to additive manufacturing (AM). Specifically, laser metal deposition (LMD) is an AM process where a laser beam melts deposited powder, and the dissolved particles fuse to produce metal components. Porosity, or small cavities that form in this printed structure, is generally considered one of the most destructive defects in metal AM. Traditionally, computer tomography scans measure porosity. While this is useful for understanding the nature of pore formation and its characteristics, purely physics-driven models lack real-time prediction ability. Meanwhile, a purely deep learning approach to porosity prediction leaves valuable physics knowledge behind. In this paper, a hybrid model that uses both empirical and simulated LMD data is created to show how various physics-informed loss functions impact the accuracy, precision, and recall of a baseline deep learning model for porosity prediction. In particular, some versions of the physics-informed model can improve the precision of the baseline deep learning-only model (albeit at the expense of overall accuracy).

Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry

Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells.

HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain's allostery and unfolding upon B12 and TonB binding

To import large metabolites across the outer membrane of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force that powers transport (1). But, the role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding (2) while others propose force-independent pore formation (3) by TonB binding leading to breakage of a salt bridge termed the "Ionic Lock". Our hydrogen exchange/mass spectrometry measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12 bound and the B12&TonB bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.

Porosity Formation in Thin Welded Joints of Al–MG–LI Alloys

This work is about the study of the correlation of pore formation in welded joints of Al–MG–LI alloy with zirconium additives with the state of the base metal, thermal vacuum treatment, and welding technologies MIG and EBW. Metallographic analysis has been carried out, the phase composition of the alloy and weld metal has been investigated, and thermal cycles of welding have been calculated, allowing to estimate the residence time of metal in the alloying zone and weld metal in the liquid state. The nature of the allocation of strengthening fine-dispersed phases in the welded joints of the alloy has been determined. The regularity and character of pore formation in welded joints depending on the applicable thermal vacuum treatment (TVT) and welding technology have been revealed. It was established that TVT with subsequent hardening and aging has no effect on the phase composition of the alloy. However, this type of treatment contributes to the formation of a more homogeneous and uniform nature of the separation of fine-dispersed strengthening phases. It was revealed that the MIG technology (metal with and without TVT) is characterized by a large length of the fusion zone, the high residence time of metal in the fusion zone and weld metal in the liquid state, and the formation of pores. Phase formation in the temperature range of the beginning and end of the alloy crystallization occurs not only in the weld at the final stage of crystallization but also in the fusion zone, which may induce pore formation, whereas EBW welding shows the opposite trend and no pores. It was found that EBW technology prevents pore formation and makes it possible to obtain welded joints of 1420 Al alloys of the required quality.