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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.

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.

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca²⁺ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca²⁺ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca²⁺ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca²⁺ fluxes.

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.

We study composites of cellulose nanocrystals (CNCs) in an ionomer matrix of poly(ethylene-stat-sodium acrylate) and find that direct cellulose/cellulose interactions in the composite are not a requirement for achieving reinforcement. While isotropic composites only show a slightly enhanced stiffness compared to the neat ionomer, a more substantial increase in Young's modulus by a factor of up to 5 is achieved by uniaxial alignment of the composites through melt spinning. The orientation of CNC in melt-spun composites reduces the probability of cellulose/cellulose interactions, which suggests that cellulose/polymer interactions must be present that lead to the observed reinforcement. Molecular dynamics simulations confirm strong cellulose/polymer interactions in the form of ionic interactions as well as hydrogen bonding. These cellulose/polymer interactions facilitate efficient stress transfer, leading to the high reinforcing effect of CNC, while cellulose/cellulose interactions play a minor role in the mechanical response of the composite.

With the growing challenges of bacteria becoming resistant to conventional antibiotics, antimicrobial peptides (AMPs) may offer a potential alternative. One of the most studied AMPs, the human cathelicidin derived AMP LL-37 is notable for its antimicrobial activity even though its mechanism of action is not fully understood yet. This work investigates the interaction of LL-37 with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) vesicles, which were employed as a bacterial membrane model given the common presence of this phospholipid in the bacterial membrane. Experimental techniques including small angle X-ray scattering, transmission electron microscopy and dynamic light scattering were used to characterize the interactions among LL-37 and POPG. Molecular dynamics simulations complement the experimental studies with molecular-level insights into the process. LL-37 was discovered to actively and critically interact with the POPG vesicles, modifying the membrane curvature that eventually leads to structural transformations from vesicles to mixed micelles. The results shed light on the mechanisms underlying the interactions among LL-37 and bacteria mimetic vesicles and can guide the further development of AMP based antimicrobial materials and therapies.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing.

The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as “peptidoglycan-binding anchor” (Pba).

Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form.