Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2-7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0-7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

A large proportion of clinical S. aureus isolates that carry an inactive Agr system are associated with persistent infection that is difficult to treat. Once S. aureus is inside the bloodstream, it can cross the endothelial barrier and invade almost every organ in the human body. Endothelial cells can either be lysed by this pathogen or they serve as a niche for its intracellular long-term survival. Following phagocytosis, several vesicles such as phagosomes and autophagosomes, target intracellular S. aureus for elimination. S. aureus can escape from these vesicles into the host cytoplasm through the activation of phenol-soluble modulins (PSMs) αβ. Thereafter, it replicates and lyses the host cell to disseminate to adjacent tissues. Herein we demonstrate that staphylococcal strains which lack the expression of PSMs employ an alternative pathway to better persist within endothelial cells. The intracellular survival of S. aureus is associated with the co-localization of the autophagy marker LC3. In cell culture infection models, we found that the absence of psmαβ decreased the host cell lysis and increased staphylococcal long-term survival. This study explains the positive selection of agr-negative strains that lack the expression of psmαβ in chronic infection due to their advantage in surviving and evading the clearance system of the host.

Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that can colonize the upper respiratory tract. It is a leading cause of a wide range of infectious diseases, including community-acquired pneumonia and meningitis. Pneumococcal infections cause 1-2 million deaths per year, most of which occur in developing countries. Here, we focused on three choline-binding proteins (CBPs), i.e., PspC, PspA, and LytA. These pneumococcal proteins have different surface-exposed regions but share related choline-binding anchors. These surface-exposed pneumococcal proteins are in direct contact with host cells and have diverse functions. We explored the role of the three CBPs on adhesion and pathogenicity in a human host by performing relevant imaging and functional analyses, such as electron microscopy, confocal laser scanning microscopy, and functional quantitative assays, targeting biofilm formation and the hemolytic capacity of S. pneumoniae. In vitro biofilm formation assays and electron microscopy experiments were used to examine the ability of knockout mutant strains lacking the lytA, pspC, or pspA genes to adhere to surfaces. We found that LytA plays an important role in robust synthesis of the biofilm matrix. PspA and PspC appeared crucial for the hemolytic effects of S. pneumoniae on human red blood cells. Furthermore, all knockout mutants caused less damage to endothelial cells than wild-type bacteria, highlighting the significance of each CPB for the overall pathogenicity of S. pneumoniae. Hence, in addition to their structural function within the cell wall of S. pneumoniae, each of these three surface-exposed CBPs controls or mediates multiple steps during bacterial pathogenesis.

Synaptic plasticity involves proper establishment and rearrangement of structural and functional microdomains. Yet, visualization of the underlying lipid cues proved challenging. Applying a combination of rapid cryofixation, membrane freeze-fracturing, immunogold labeling and electron microscopy, we visualize and quantitatively determine the changes and the distribution of phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane of dendritic spines and subareas thereof at ultra-high resolution. These efforts unravel distinct phases of PIP2 signals during induction of long-term depression (LTD). During the first minutes PIP2 rapidly increases in a PIP5K-dependent manner forming nanoclusters. PTEN contributes to a second phase of PIP2 accumulation. The transiently increased PIP2 signals are restricted to upper and middle spine heads. Finally, PLC-dependent PIP2 degradation provides timely termination of PIP2 cues during LTD induction. Together, this work unravels the spatial and temporal cues set by PIP2 during different phases after LTD induction and dissects the molecular mechanisms underlying the observed PIP2 dynamics.

We report the x-ray crystal structure of intact, full-length human immunoglobulin (IgG4) at 1.8 Å resolution. The data for IgG4 (S228P), an antibody targeting the natriuretic peptide receptor A, show a previously unrecognized type of Fab-Fc orientation with a distorted λ-shape in which one Fab-arm is oriented toward the Fc portion. Detailed structural analysis by x-ray crystallography and molecular simulations suggest that this is one of several conformations coexisting in a dynamic equilibrium state. These results were confirmed by small angle x-ray scattering in solution. Furthermore, electron microscopy supported these findings by preserving molecule classes of different conformations. This study fosters our understanding of IgG4 in particular and our appreciation of antibody flexibility in general. Moreover, we give insights into potential biological implications, specifically for the interaction of human anti-natriuretic peptide receptor A IgG4 with the neonatal Fc receptor, Fcγ receptors, and complement-activating C1q by considering conformational flexibility.

The intestine is the primary reservoir of Candida albicans that can cause systemic infections in immunocompromised patients. In this reservoir, the fungus exists as a harmless commensal. However, antibiotic treatment can disturb the bacterial microbiota, facilitating fungal overgrowth and favoring pathogenicity. The current in vitro gut models that are used to study the pathogenesis of C. albicans investigate the state in which C. albicans behaves as a pathogen rather than as a commensal. We present a novel in vitro gut model in which the fungal pathogenicity is reduced to a minimum by increasing the biological complexity. In this model, enterocytes represent the epithelial barrier and goblet cells limit C. albicans adhesion and invasion. Significant protection against C. albicans-induced necrotic damage was achieved by the introduction of a microbiota of antagonistic lactobacilli. We demonstrated a time-, dose- and species-dependent protective effect against C. albicans-induced cytotoxicity. This required bacterial growth, which relied on the presence of host cells, but was not dependent on the competition for adhesion sites. Lactobacillus rhamnosus reduced hyphal elongation, a key virulence attribute. Furthermore, bacterial-driven shedding of hyphae from the epithelial surface, associated with apoptotic epithelial cells, was identified as a main and novel mechanism of damage protection. However, host cell apoptosis was not the driving mechanism behind shedding. Collectively, we established an in vitro gut model that can be used to experimentally dissect commensal-like interactions of C. albicans with a bacterial microbiota and the host epithelial barrier. We also discovered fungal shedding as a novel mechanism by which bacteria contribute to the protection of epithelial surfaces.This article has an associated First Person interview with the joint first authors of the paper.

The self-assembly of Aβ peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer's disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments.

Apolipoprotein-E (ApoE) has been implicated in Alzheimer's disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (KD~140-580 pM) in vitro, and C1q-ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aβ plaques, and atherosclerosis in vivo. C1q-ApoE complexes in human ChPs, Aβ plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aβ-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.

Establishment of multicellularity represents a major transition in eukaryote evolution. A subgroup of Amoebozoa, the dictyosteliids, has evolved a relatively simple aggregative multicellular stage resulting in a fruiting body supported by a stalk. Protosteloid amoeba, which are scattered throughout the amoebozoan tree, differ by producing only one or few single stalked spores. Thus, one obvious difference in the developmental cycle of protosteliids and dictyosteliids seems to be the establishment of multicellularity. To separate spore development from multicellular interactions, we compared the genome and transcriptome of a Protostelium species (Protostelium aurantium var. fungivorum) with those of social and solitary members of the Amoebozoa. During fruiting body formation nearly 4,000 genes, corresponding to specific pathways required for differentiation processes, are upregulated. A comparison with genes involved in the development of dictyosteliids revealed conservation of >500 genes, but most of them are also present in Acanthamoeba castellanii for which fruiting bodies have not been documented. Moreover, expression regulation of those genes differs between P. aurantium and Dictyostelium discoideum. Within Amoebozoa differentiation to fruiting bodies is common, but our current genome analysis suggests that protosteliids and dictyosteliids used different routes to achieve this. Most remarkable is both the large repertoire and diversity between species in genes that mediate environmental sensing and signal processing. This likely reflects an immense adaptability of the single cell stage to varying environmental conditions. We surmise that this signaling repertoire provided sufficient building blocks to accommodate the relatively simple demands for cell-cell communication in the early multicellular forms.

Previous studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model.

Microbial communities involving dehalogenating bacteria assist in bioremediation of areas contaminated with halocarbons. To understand molecular interactions between dehalogenating bacteria, we co-cultured Sulfurospirillum multivorans, dechlorinating tetrachloroethene (PCE) to cis-1,2-dichloroethene (cDCE), and Dehalococcoides mccartyi strains BTF08 or 195, dehalogenating PCE to ethene. The co-cultures were cultivated with lactate as electron donor. In co-cultures, the bacterial cells formed aggregates and D. mccartyi established an unusual, barrel-like morphology. An extracellular matrix surrounding bacterial cells in the aggregates enhanced cell-to-cell contact. PCE was dehalogenated to ethene at least three times faster in the co-culture. The dehalogenation was carried out via PceA of S. multivorans, and PteA (a recently described PCE dehalogenase) and VcrA of D. mccartyi BTF08, as supported by protein abundance. The co-culture was not dependent on exogenous hydrogen and acetate, suggesting a syntrophic relationship in which the obligate hydrogen consumer D. mccartyi consumes hydrogen and acetate produced by S. multivorans. The cobamide cofactor of the reductive dehalogenase-mandatory for D. mccartyi-was also produced by S. multivorans. D. mccartyi strain 195 dechlorinated cDCE in the presence of norpseudo-B12 produced by S. multivorans, but D. mccartyi strain BTF08 depended on an exogenous lower cobamide ligand. This observation is important for bioremediation, since cofactor supply in the environment might be a limiting factor for PCE dehalogenation to ethene, described for D. mccartyi exclusively. The findings from this co-culture give new insights into aggregate formation and the physiology of D. mccartyi within a bacterial community.

'Candidatus Streptomyces philanthi' is a monophyletic clade of formerly uncultured bacterial symbionts in solitary digger wasps of the genera Philanthus, Philanthinus and Trachypus (Hymenoptera, Crabronidae). These bacteria grow in female-specific antennal reservoirs and - after transmission to the cocoon - produce antibiotics protecting the host larvae from fungal infection. However, the symbionts' refractoriness to cultivation has thus far hampered detailed in vitro studies on their physiology and on the evolutionary changes in metabolic versatility in response to the host environment.

Upon systemic infection with human pathogenic yeast Candida albicans (C. albicans), human monocytes and polymorph nuclear neutrophilic granulocytes are the first immune cells to respond and come into contact with C. albicans. Monocytes exert immediate candidacidal activity and inhibit germination, mediate phagocytosis, and kill fungal cells. Here, we show that human monocytes spontaneously respond to C. albicans cells via phagocytosis, decondensation of nuclear DNA, and release of this decondensed DNA in the form of extracellular traps (called monocytic extracellular traps: MoETs). Both subtypes of monocytes (CD14++CD16-/CD14+CD16+) formed MoETs within the first hours upon contact with C. albicans. MoETs were characterized by the presence of citrullinated histone, myeloperoxidase, lactoferrin, and elastase. MoETs were also formed in response to Staphylococcus aureus and Escherichia coli, indicating a general reaction of monocytes to infectious microbes. MoET induction differs from extracellular trap formation in macrophages as MoETs are not triggered by simvastatin, an inhibitor of cholesterol synthesis and inducer of extracellular traps in macrophages. Extracellular traps from both monocytes and neutrophils activate complement and C3b is deposited. However, factor H (FH) binds via C3b to the extracellular DNA, mediates cofactor activity, and inhibits the induction of the inflammatory cytokine interleukin-1 beta in monocytes. Altogether, the results show that human monocytes release extracellular DNA traps in response to C. albicans and that these traps finally bind FH via C3b to presumably support clearance without further inflammation.

Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests.

The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood.

The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-β1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble β-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-β1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-β1 to the TGF-β receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-β1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-β1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.

Zinc oxide nanoparticles (ZnO NP) offer beneficial properties for many applications, especially in the food sector. Consequently, as part of the human food chain, they are taken up orally. The toxicological evaluation of orally ingested ZnO NP is still controversial. In addition, their physicochemical properties can change during digestion, which leads to an altered biological behaviour. Therefore, the aim of our study was to investigate the fate of two different sized ZnO NP (< 50 nm and < 100 nm) during in vitro digestion and their effects on model systems of the intestinal barrier. Differentiated Caco-2 cells were used in mono- and coculture with mucus-producing HT29-MTX cells. The cellular uptake, the impact on the monolayer barrier integrity and cytotoxic effects were investigated after 24 h exposure to 123-614 µM ZnO NP.