Internalization and toxicity of polystyrene nanoplastics on inmortalized human neural stem cells.

Global plastic production has increased exponentially in recent decades, and a significant part of it persists in the environment, where it degrades into microplastics and nanoplastics (MPs and NPs). These can enter in humans by ingestion, inhalation, and dermal routes, and there is scientific evidence that they are able to reach the systemic circulation and penetrate and accumulate in various tissues and organs. Neurodevelopmental toxicity of NPs is one of the most worrying effects, as they can cross the blood-brain barrier. In the following study, we analyzed, by transmission electron microscopy, the in vitro uptake of 30-nm polystyrene nanoplastics (PS-NPs) into human neural stem cells (NSCs), their accumulation and subcellular localization within the cell. Furthermore, we studied the effects of different concentrations of PS-NPs on cell death, proliferation, and cell differentiation using immunocytochemistry and quantitative real time PCR for specific markers. This study demonstrated that PS-NPs were able to enter the cell, probably by endocytosis, accumulate, and aggregated in human NSCs, without being detected in the nucleus, causing cell death by apoptosis and decreased cell proliferation. This study provides new insights into the interaction and effects of PS-NPs in human NSC and supports the scientific evidence for the involvement of nanoplastic in neurodevelopmental disorders.

Accumulation of endogenous free radicals is required to induce titan-like cell formation in Cryptococcus neoformans.

IMPORTANCE: Cryptococcus neoformans is an excellent model to investigate fungal pathogenesis. This yeast can produce "titan cells," which are cells of an abnormally larger size that contribute to the persistence of the yeast in the host. In this work, we have used a new approach to characterize them by identifying drugs that inhibit this process. We have used a repurposing off-patent drug library, combined with an automatic method to image and analyze fungal cell size. In this way, we have identified many compounds that inhibit this transition. Interestingly, several compounds were antioxidants, allowing us to confirm that endogenous ROS and mitochondrial changes are important for titan cell formation. This work provides new evidence of the mechanisms required for titanization. Furthermore, the future characterization of the inhibitory mechanisms of the identified compounds by the scientific community will contribute to better understand the role of titan cells in virulence.

Middle East respiratory coronavirus (MERS-CoV) internalized by llama alveolar macrophages does not result in virus replication or induction of pro-inflammatory cytokines.

Severe Middle East respiratory syndrome (MERS) is characterized by massive infiltration of immune cells in lungs. MERS-coronavirus (MERS-CoV) replicates in vitro in human macrophages, inducing high pro-inflammatory responses. In contrast, camelids, the main reservoir for MERS-CoV, are asymptomatic carriers. Although limited infiltration of leukocytes has been observed in the lower respiratory tract of camelids, their role during infection remains unknown. Here we studied whether llama alveolar macrophages (LAMs) are susceptible to MERS-CoV infection and can elicit pro-inflammatory responses. MERS-CoV did not replicate in LAMs; however, they effectively capture and degrade viral particles. Moreover, transcriptomic analyses showed that LAMs do not induce pro-inflammatory cytokines upon MERS-CoV sensing.

Molecular mapping of virus-infected cells with immunogold and metal-tagging transmission electron microscopy.

Transmission electron microscopy (TEM) has been essential to study virus-cell interactions. The architecture of viral replication factories, the principles of virus assembly and the components of virus egress pathways are known thanks to the contribution of TEM methods. Specially, when studying viruses in cells, methodologies for labeling proteins and other macromolecules are important tools to correlate morphology with function. In this review, we present the most widely used labeling method for TEM, immunogold, together with a lesser known technique, metal-tagging transmission electron microscopy (METTEM) and how they can contribute to study viral infections. Immunogold uses the power of antibodies and electron dense, colloidal gold particles while METTEM uses metallothionein (MT), a metal-binding protein as a clonable tag. MT molecules build gold nano-clusters inside cells when these are incubated with gold salts. We describe the necessary controls to confirm that signals are specific, the advantages and limitations of both methods, and show some examples of immunogold and METTEM of cells infected with viruses.

ß-Cyclodextrins as affordable antivirals to treat coronavirus infection.

The SARS-CoV-2 pandemic made evident that there are only a few drugs against coronavirus. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety profile. Starting from a list of 116 drug candidates, we used molecular modelling tools to rank the 44 most promising inhibitors. Next, we tested their efficacy as antivirals against α and ß coronaviruses, such as the HCoV-229E and SARS-CoV-2 variants. Four drugs, OSW-1, U18666A, hydroxypropyl-ß-cyclodextrin (HßCD) and phytol, showed in vitro antiviral activity against HCoV-229E and SARS-CoV-2. The mechanism of action of these compounds was studied by transmission electron microscopy and by fusion assays measuring SARS-CoV-2 pseudoviral entry into target cells. Entry was inhibited by HßCD and U18666A, yet only HßCD inhibited SARS-CoV-2 replication in the pulmonary Calu-3 cells. Compared to the other cyclodextrins, ß-cyclodextrins were the most potent inhibitors, which interfered with viral fusion via cholesterol depletion. ß-cyclodextrins also prevented infection in a human nasal epithelium model ex vivo and had a prophylactic effect in the nasal epithelium of hamsters in vivo. All accumulated data point to ß-cyclodextrins as promising broad-spectrum antivirals against different SARS-CoV-2 variants and distant alphacoronaviruses. Given the wide use of ß-cyclodextrins for drug encapsulation and their high safety profile in humans, our results support their clinical testing as prophylactic antivirals.

The SNARE protein SNAP-25 is required for normal exocytosis at auditory hair cell ribbon synapses.

Hearing depends on fast and sustained calcium-dependent synaptic vesicle fusion at the ribbon synapses of cochlear inner hair cells (IHCs). The implication of the canonical neuronal SNARE complex in this exocytotic process has so far remained controversial. We investigated the role of SNAP-25, a key component of this complex, in hearing, by generating and analyzing a conditional knockout mouse model allowing a targeted postnatal deletion of Snap-25 in IHCs. Mice subjected to IHC Snap-25 inactivation after hearing onset developed severe to profound deafness because of defective IHC exocytosis followed by ribbon degeneration and IHC loss. Viral transfer of Snap-25 in these mutant mice rescued their hearing function by restoring IHC exocytosis and preventing synapses and hair cells from degeneration. These results demonstrate that SNAP-25 is essential for normal hearing function, most likely by ensuring IHC exocytosis and ribbon synapse maintenance.

A protocol for ultrastructural study of Candida albicans biofilm using transmission electron microscopy.

This protocol describes how to analyze C. albicans biofilm using transmission electron microscopy. We present two approaches to observe the ultrastructure of fungal cells within unperturbed biofilms, as well as an immunogold labeling procedure. This approach maintains the architecture of the fungal biofilm close to its native state by growing C. albicans biofilm on a plastic surface. After the freeze substitution procedure, classical transmission electron microscopy or electron tomography will allow the ultrastructural analysis of the microbial community.

C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus.

The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis.

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-ß-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles from endosomes into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

Unraveling the antiviral activity of plitidepsin against SARS-CoV-2 by subcellular and morphological analysis.

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation elongation factor 1 alpha) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner.

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals.

The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus-host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus-host interactions, we studied the life cycle of the enveloped, ∼2-µm-long Sulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus-host interactions in Archaea.

High-throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts.

Mutations in OPA1 cause autosomal dominant optic atrophy (DOA) as well as DOA+, a phenotype characterized by more severe neurological deficits. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae, respiration, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. We screened the entire known mitochondrial proteome (1,531 genes) to identify genes that control mitochondrial morphology using a first-in-kind imaging pipeline. We identified 145 known and novel candidate genes whose depletion promoted elongation or fragmentation of the mitochondrial network in control fibroblasts and 91 in DOA+ patient fibroblasts that prevented mitochondrial fragmentation, including phosphatidyl glycerophosphate synthase (PGS1). PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1-deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology, or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation.

Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria.

Bacteria synthesize a wide range of intracellular submicrometer-sized inorganic precipitates of diverse chemical compositions and structures, called biominerals. Their occurrences, functions and ultrastructures are not yet fully described despite great advances in our knowledge of microbial diversity. Here, we report bacteria inhabiting the sediments and water column of the permanently stratified ferruginous Lake Pavin, that have the peculiarity to biomineralize both intracellular magnetic particles and calcium carbonate granules. Based on an ultrastructural characterization using transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), we showed that the calcium carbonate granules are amorphous and contained within membrane-delimited vesicles. Single-cell sorting, correlative fluorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and molecular typing of populations inhabiting sediments affiliated these bacteria to a new genus of the Alphaproteobacteria. The partially assembled genome sequence of a representative isolate revealed an atypical structure of the magnetosome gene cluster while geochemical analyses indicate that calcium carbonate production is an active process that costs energy to the cell to maintain an environment suitable for their formation. This discovery further expands the diversity of organisms capable of intracellular Ca-carbonate biomineralization. If the role of such biomineralization is still unclear, cell behaviour suggests that it may participate to cell motility in aquatic habitats as magnetite biomineralization does.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

The viral replication organelles within cells studied by electron microscopy.

Transmission electron microscopy (TEM) has been crucial to study viral infections. As a result of recent advances in light and electron microscopy, we are starting to be aware of the variety of structures that viruses assemble inside cells. Viruses often remodel cellular compartments to build their replication factories. Remarkably, viruses are also able to induce new membranes and new organelles. Here we revise the most relevant imaging technologies to study the biogenesis of viral replication organelles. Live cell microscopy, correlative light and electron microscopy, cryo-TEM, and three-dimensional imaging methods are unveiling how viruses manipulate cell organization. In particular, methods for molecular mapping in situ in two and three dimensions are revealing how macromolecular complexes build functional replication complexes inside infected cells. The combination of all these imaging approaches is uncovering the viral life cycle events with a detail never seen before.

Aspergillus fumigatus exoß(1-3)glucanases family GH55 are essential for conidial cell wall morphogenesis.

The cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched ß(1-3)glucan, to which the chitin and the galactomannan are covalently bound. Softening of the cell wall is an essential event during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosyl hydrolases. In this study, we characterised the role of the glycosyl hydrolase GH55 members in A. fumigatus fungal morphogenesis. We showed that deletion of the six genes of the GH55 family stopped conidial cell wall maturation at the beginning of the development process, leading to abrogation of conidial separation: the shape of conidia became ovoid, and germination was delayed. In conclusion, the reorganisation and structuring of the conidial cell wall mediated by members of the GH55 family is essential for their maturation, normal dissemination, and germination.

Listeriolysin O-dependent host surfaceome remodeling modulates Listeria monocytogenes invasion.

Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.