Biology subjects

Sandblad, L.

Publications and source records attributed to Sandblad, L..

8 recordsLinked to original sources

bioRxiv2025

Uromodulin velcro sheets and their interaction with uropathogenic E. coli

Homopolymeric uromodulin (UMOD), the most abundant protein in human urine, protects against urinary tract infections (UTI) by acting as a decoy for uropathogenic E. coli (UPEC). Using cryo-electron tomography (ET), we show that urinary UMOD filaments naturally form sheets that interact with UPEC type I pili. Sheet formation is salt-dependent, and we resolve their high-resolution structure using single-particle cryo-electron microscopy (EM). This reveals a lateral interface between polymers, whose mutation disrupts UMOD filament bundle formation in mammalian cells. Branchless egg coat protein ZPD also forms sheets, and cryo-ET of elastase-treated UMOD indicates that absence of N-terminal branches promotes the stacking of UMOD-like protein sheets into thick 3D matrices. These results rationalize early observations of salt-dependent aggregation of UMOD, explain how UMOD can assemble into extended velcro-like structures that efficiently inactivate a multitude of adhesive UPEC pili, and suggest how UMOD-like molecules can generally organize into supramolecular structures of variable thickness.

biophysics↗

bioRxiv2025

Bacteroidales T6SS minor Hcp subunits form heteromers recognising effectors

The type VI secretion system (T6SS) is a macromolecular protein complex found in Gram-negative bacteria that mediates intercellular antagonism. T6SSs are gut colonization factors that influence gut biodiversity. 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 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 analyses demonstrated that Hcp1 and Hcp2 assemble into heterohexamers and that this complex recognizes the secreted effector Bte1. 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.

biochemistry↗

bioRxiv2024

Combining live cell fluorescence imaging with in situ cryo electron tomography sheds light on the septation process in Deinococcus radiodurans

Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes, but 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 super-resolution 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 of the cell envelope of this bacterium, (ii) the sliding doors septation process and (iii) the molecular mechanisms underlying septal growth and closure.

microbiology↗

bioRxiv2024

Nutrient-deprived growth of streptomycetes reveals a novel growth phenotype with enhanced antimicrobial activity

Species of the genus Streptomyces are filamentous bacteria mainly residing in soils. The capacity of streptomycetes to adapt to various environments is reflected in the sheer size of their genomes and the number of encoded regulatory proteins. The nutrient-rich laboratory conditions used for culturing significantly differ from the conditions in the natural habitat of streptomycetes and fail to stimulate their full range of adaptive behaviors; as on of the results, the majority of biosynthetic gene clusters remain silent. Moreover, the use of rich media has led to the assumption that nutrient depletion ultimately promotes sporulation. However, we demonstrate that S. coelicolor colonies can respond to nutrient depletion by displaying a previously unidentified phenotypic transition termed "foraging" due to its submerged and continuous growth on depleted media. The foraging phenotype is distinctly different from conventional phenotypes in terms of colony morphology, genomic stability, and metabolomic profile. This adaptation to nutrient deprivation through foraging is found throughout the Streptomyces phylogeny, indicating that the phenotype is highly conserved. Furthermore, foraging S. coelicolor gains the ability to inhibit molds and enhanced competitive activity against both gram-negative and gram-positive bacteria is detected in other species. These findings highlight how morphological adaptations of streptomycetes in nutrient-limited environments alter secondary metabolite production, enabling the screening of novel antimicrobial activities. These discoveries have implications ranging from the basic biology of streptomycetes to drug discovery and microbial ecology.

microbiology↗

bioRxiv2024

Exosomes are vehicles for the stress-regulated secretion of histones

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 histone EV 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 or histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. EV histones colocalized most frequently with the tetraspanin CD63 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.

cell biology↗

bioRxiv2023

Bile acids accumulate norovirus-like particles and enhance binding to and entry into human enteric epithelial cells

Human norovirus (HuNoV) is a leading cause of acute viral gastroenteritis, but despite high impact on public health and health care, the mechanisms of viral attachment to and entry into target cells are not yet fully understood. It is well known that body fluids such as blood can transmit unrelated viruses, but recent reports also indicate that saliva and bile contribute to 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 accumulation of viral particles in the vicinity of cells. Our 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. ImportanceViruses transmitted by the fecal-oral route encounter secreted host factors in gastrointestinal fluids. Some host factors can be exploited by the virus to facilitate infection. Human bile acids indirectly promote norovirus entry into and infection of human enteroids, but the direct effect of bile acids on attachment and uptake, along with the impact of other gastrointestinal fluids, remain unknown. Here, we investigated the direct effects of human body fluids on cellular attachment of norovirus VLPs. We show that human bile and hydrophobic bile acids induce an accumulation of norovirus VLPs, which is associated with significantly enhanced attachment and entry into human duodenal cell lines. These results highlight the differential effects of gastrointestinal body fluids on viral attachment and entry, while providing useful information into the complex HuNoV-host interactions that facilitate infection.

microbiology↗

bioRxiv2021

Cytoskeleton-extracellular matrix interaction is required to maintain mitochondrial Ca2+ control in mouse skeletal muscle fibre

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. Here, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling in adult mouse skeletal muscle fibers. Adult skeletal muscle fibers were isolated from mouse toe muscle either by collagenase-induced dissociation of the ECM or by mechanical dissection that leaves the contiguous ECM intact. Experiments were generally performed four hours after cell isolation. At this time, there were striking differences in the gene expression patterns between fibers isolated with the two methods; 24h after cell isolation, enzymatically dissociated fibers had transcriptomic signatures resembling dystrophic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria in enzymatically dissociated than in mechanically dissected fibers. Similar increases in free cytosolic [Ca2+] during repeated tetanic stimulation were accompanied by marked mitochondrial Ca2+ uptake only in enzymatically dissociated muscle fibers. The aberrant mitochondrial Ca2+ uptake was partially prevented by the mitochondrial Ca2+ uniporter inhibitor Ru360 and by cyclosporine A and NV556, which inhibit the mitochondrial protein Ppif (also called cyclophilin D). Importantly, inhibition of Ppif with NV556 significantly improved survival of mice with mitochondrial myopathy in which muscle mitochondria take up excessive amounts of Ca2+ even with an intact ECM. In conclusion, skeletal muscle fibers isolated by collagenase-induced dissociation of the ECM display aberrant mitochondrial Ca2+ uptake, which involves a Ppif-dependent mitochondrial Ca2+ influx resembling that observed in mitochondrial myopathies.

physiology↗

bioRxiv2018

DSeg: A dynamic image segmentation program to extract backbone patterns for filamentous bacteria and hyphae structures

MotivationQuantitative image analysis of growing filamentous fungi and prokaryotes are important to detect and evaluate morphological effects of growth conditions, compounds and mutations. However, analysis of time-series image data is often limited by the ability of the algorithms to accurately segment structures that are complicated or if an organism is within a crowded population. To overcome these issues we present DSeg; an image analysis program designed to process time-series image data as well as single images to find multiple filamentous structures e.g., filamentous prokaryotes, yeasts and molds using a dynamic segmentation approach. DSeg automatically segments and analyzes objects, includes drift correction, and outputs statistical data such as persistence length, growth rate and growth direction.\n\nAvailability and implementationDSeg is a free open-source program written in MATLAB. DSeg can be downloaded as a package from https://sourceforge.net/projects/dseg-software.\n\nContactmagnus.andersson@umu.se and hanqing.zhang@umu.se\n\nSupplementary informationSupplementary data are available at online.

bioinformatics↗