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Human norovirus (HuNoV) is a leading cause of acute viral gastroenteritis, but despite high impact on public health and healthcare, the mechanisms of viral attachment to and entry into target cells are not yet fully understood. Recent reports indicate that saliva and bile contribute to the transmission of HuNoV. For example, human bile acids increase cell surface ceramide levels in human enteroids, which improves norovirus entry into cells, resulting in enhanced replication. Bile acids can also interact directly with the norovirus capsid, but it is not known whether bile or other gastrointestinal body fluids directly affect HuNoV attachment to host cells. In this study, we investigated the effects of patient-derived gastric juice, pancreatic juice, and bile on HuNoV GII.4 virus-like particle (VLP) attachment to and entry into a human duodenal cell line, HuTu-80. We show that while gastric juice and pancreatic juice do not affect viral attachment or entry, bile—in particular, hydrophobic bile acids—significantly enhance cellular attachment and subsequent entry of GII.4 VLPs into cells. In addition, we show that hydrophobic bile acids induce the accumulation of viral particles in the vicinity of cells. These results suggest the presence of a new en masse infection mechanism, where bile acids aggregate virions and allow direct and more efficient attachment to and entry into target cells.

Unicellular organisms or cells of metazoans often change their morphology during development or life cycle progression to adapt to environmental changes. Malaria parasites undergo a striking range of morphological transformations as they navigate through the different environments of mammalian hosts and mosquito vectors. These developmental transitions are accompanied by changes in the subcellular organelles. Here, this work introduces an unbiased approach using volume electron microscopy (vEM) to facilitate cluster analyses of morphometric parameters during developmental transformation. Investigating the transformation of fertilized Plasmodium zygotes into the motile ookinetes with three complementary vEM techniques revealed intimate mitochondrion-nucleus interactions, different microtubule arrangements, elongated shapes of micronemes and their close interaction with the apicoplast. The presented data and approach provide an open-access subcellular atlas for ookinete development to aid mechanistic molecular insights from reverse genetic studies and a framework for the ultrastructural study of other parasite stages and developmental transitions in general.

The Encephaliozoon cuniculi is an obligate intracellular microsporidian parasite with a highly reduced genome, yet it contains several key components of an actin cytoskeleton. In this study, we characterise the α-actinin-like protein from the parasite to gain insight into its role in actin organisation. The protein contains three domains typical of α-actinins: an N-terminal actin-binding domain and a C-terminal calmodulin-like domain, separated by a rod domain. Gel filtration analysis demonstrated that the recombinant protein formed stable dimers, consistent with the canonical antiparallel α-actinin structure. Actin co-sedimentation assays and electron microscopy confirmed that the α-actinin-like protein binds and cross-links actin filaments into tight bundles, whereas the isolated actin-binding domain binds but does not cross-link filaments. AlphaFold modelling predicted an overall structural arrangement compatible with an antiparallel dimer. Our results identify the E. cuniculi α-actinin-like protein as a true α-actinin homologue. The presence of actin-binding proteins in E. cuniculi as well as in other microsporidia with very small genomes implies that an actin-based cytoskeleton is important for their survival.

Multidrug-resistant Gram-negative bacteria are a growing clinical threat, driving the search for alternative antimicrobial strategies, such as antimicrobial peptide (AMP)- based materials. However, the rational design of such systems remains constrained by simplified membrane models that neglect critical components of the Gram-negative envelope, such as lipopolysaccharides and cardiolipin, and fail to capture its dual-membrane architecture. This work establishes a materials-oriented experimental framework for constructing membrane-mimetic oligolamellar structures that actively integrate the human AMP LL-37. These hierarchically organized assemblies emulate the compositional and structural complexity of the Gram-negative inner and outer membranes and have the potential to serve as tunable soft-matter platforms for the delivery of AMPs. Combining small-angle X-ray scattering, electron microscopy, electrophoretic mobility analysis, and coarse-grained molecular dynamics simulations, we show that LL-37 interacts strongly with cardiolipin, driving phase transitions from multilamellar vesicles to nanoscale assemblies, followed by membrane stacking. This restructuring phenomenon is unlikely to occur in conventional single-bilayer systems. In the presence of lipopolysaccharides, polysaccharide side chains modulate but do not suppress this transition, revealing a lipid-specific reorganisation mechanism relevant to the design of AMP-based materials targeting Gram-negative bacteria. These results deepen mechanistic understanding of AMP-membrane interactions and establish design principles for peptide-integrated soft materials with programmable structural responses. The presented platform further enables the development of antimicrobial biointerfaces through targeted membrane remodeling.

The type VI secretion system (T6SS) is a protein complex found in Gram-negative bacteria that mediates intercellular antagonism. Hemolysin-coregulated proteins (Hcp) are major structural proteins in these systems. Hcp forms hexameric rings that stack to create an inner tube structure essential for translocating effector proteins into target cells. In gut Bacteroidales, T6SS loci encode multiple Hcp proteins with unknown function. The gut commensal Bacteroides fragilis encodes five Hcp subunits (sHcp and Hcp1-4) that have low sequence similarity. In this study, we investigated the roles of these proteins. Interaction studies showed that sHcp forms homohexamers, which is consistent with a major role of forming the bulk of the inner tube. In contrast, the less abundant minor Hcp1-4 were shown to form an interaction network involving heteromeric complexes. Biochemical and genetic analyses demonstrated that Hcp1 and Hcp2 assemble into heterohexamers and that this complex recognizes the secreted effector Bte1, contributing to its secretion. Finally, we showed that Hcp modules, which are encoded in highly syntenic regions in T6SS loci of Bacteroidales, cluster with effectors. These results imply that the minor Hcps genetically cosegregate with cognate effectors, contributing to effector cassette variability. Thus, minor Hcp subunits function as recognition particles for effectors to mediate secretion, which appears to be a conserved trait in Bacteroidales T6SSs. Exploiting these features could facilitate the characterization of unknown effectors by copurifying them with their cognate Hcps. This approach may reveal new insights into bacterial interactions and the mechanisms that establish gut biodiversity.

Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In walled bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes. In all cases, division involves a step of septation, corresponding to the growth of a new dividing cell wall, followed by splitting of the two daughter cells. The radiation-resistant Deinococcus radiodurans is a spherical bacterium protected by a thick and unusual cell envelope. It has been reported to divide using a distinctive mode of septation in which two septa originating from opposite sides of the cell progress with a flat leading edge until meeting and fusing at mid-cell. In the present study, we have combined conventional and superresolution fluorescence microscopy of live bacteria with in situ cryogenic electron tomography of bacterial lamellae to investigate the septation process in D. radiodurans. This work provides important insight into i) the complex architecture and multilayered composition of the cell envelope of this bacterium, ii) the unusual "sliding doors" septation process and iii) the sequence of events and molecular mechanisms underlying septal closure, including the synthesis of a FtsZ-dependent peptidoglycan layer that rigidifies and straightens the growing septa.

Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals, and when elevated, may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of EV histone secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones  and histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. Histones colocalized to tetraspanin positive EVs but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease.

The human body employs nutritional immunity to restrict essential micronutrients, such as zinc, from invading pathogens, impeding their growth and replication. Here, we applied an advanced nanochemical imaging technique, synchrotron radiation-based X-ray fluorescence (SR-XRF), on vitrified polymorphonuclear neutrophils (PMNs) during the occurrence of phagocytosis of Saccharomyces cerevisiae. Nanoscopic SR-XRF provided trace elemental distributions at 50 nm spatial resolution, revealing the metal interplay between PMNs and S. cerevisiae. Our results were complemented with X-ray holographic nanotomography (XNH), confirming phagocytosis and providing complementary intracellular morphological information. A systematic decrease in zinc was observed between free and phagocytosed S. cerevisiae within the same XRF maps, suggesting active zinc depletion by PMNs. Other elements, such as sulfur, show an increase in the phagosome, likely indicative of the increase in proteins in the vicinity of phagocytic events. Through 2D/3D nanoimaging and time-lapse microscopy, we confirmed the reduction of zinc within phagocytosed yeast. Hence, our findings challenge the currently accepted hypothetical model that PMNs intoxicate engulfed microbes with an overwhelming influx of zinc ions into the phagosome. Furthermore, antimicrobial assays demonstrated that S. cerevisiae can cope well with sudden zinc spikes. Even high zinc concentrations imposed on S. cerevisiae grown under zinc-limiting conditions did not have adverse effects on viability. Contrarily, S. cerevisiae was more resistant to phagocytic killing by PMNs when grown under high zinc concentrations before infection. Our findings further consolidate zinc deprivation as an effective antimicrobial strategy. A better understanding of the metal deprivation mechanisms could inspire new exploitable targets for antimicrobial therapies.

Prokaryotic maintenance respiration and associated metabolic activities constitute a considerable proportion of the total respiration of carbon to CO2 in the ocean's mixed layer. However, seasonal influences on prokaryotic maintenance activities in terms of morphological and metabolic adaptations at low (winter) and high productivity (summer) are still unclear. To address this, we examined the natural prokaryotic communities at the mesocosm scale to analyse the differences in their morphological features and gene expression at low and high maintenance respiration, experimentally manipulated with the specific growth rate. Here, we showed that morphological features including membrane blebbing, membrane vesicles, and cell-cell connections occurred under high productivity. Metabolic adaptations associated with maintenance activities were observed under low productivity. Several Kyoto Encyclopedia of Genes and Genomes categories related to signal transduction, energy metabolism, and translational machinery supported maintenance activities under simulated winter conditions. Differential abundances of genes related to transporters, osmoregulation, nitrogen metabolism, ribosome biogenesis, and cold stress were observed. Our results demonstrate how specific growth rate in different seasons can influence resource allocation at the levels of morphological features and metabolic adaptations. This motivates further study of morphological features and their ecological role during high productivity, while investigations of metabolic adaptations during low productivity can advance our knowledge about maintenance activities.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5^CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5^CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5^CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.