Flanking Domains Modulate α-Synuclein Monomer Structure: A Molecular Dynamics Domain Deletion Study.

Aggregates of misfolded α-synuclein proteins (asyn) are key markers of Parkinson's disease. Asyn proteins have three domains: an N-terminal domain, a hydrophobic NAC core implicated in aggregation, and a proline-rich C-terminal domain. Proteins with truncated C-terminal domains are known to be prone to aggregation and suggest that studying domain-domain interactions in asyn monomers could help elucidate the role of the flanking domains in modulating protein structure. To this end, we used Gaussian accelerated molecular dynamics (GAMD) to simulate wild-type (WT), N-terminal truncated (DN), C-terminal truncated (ΔC), and isolated NAC domain variants (isoNAC). Using clustering and contact analysis, we found that N- and C-terminal domains interact via electrostatic interactions, while the NAC and N-terminal domains interact through hydrophobic contacts. Our work also suggests that the C-terminal domain does not interact directly with the NAC domain but instead interacts with the N-terminal domain. Removal of the N-terminal domain led to increased contacts between NAC and C-terminal domains and the formation of interdomain ß-sheets. Removal of either flanking domain also resulted in increased compactness of every domain. We also found that the contacts between flanking domains results in an electrostatic potential (ESP) that could possibly lead to favorable interactions with anionic lipid membranes. Removal of the C-terminal domain disrupts the ESP in a way that is likely to over-stabilize protein-membrane interactions. All of this suggests that one of the roles of the flanking domains may be to modulate the protein structure in a way that helps maintain elongation, hide hydrophobic residue from the solvent, and maintain an ESP that aids favorable interactions with the membrane.

Chitinase-like proteins promoting tumorigenesis through disruption of cell polarity via enlarged endosomal vesicles.

Introduction: Chitinase-like proteins (CLPs) are associated with tissue-remodeling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, CLP's role in tumors is far from clear. Methods: Here, we utilize Drosophila melanogaster and molecular genetics to investigate the function of CLPs (imaginal disc growth factors; Idgf's) in RasV12 dysplastic salivary glands. Results and discussion: We find one of the Idgf's members, Idgf3, is transcriptionally induced in a JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EnVs) that promote tumor progression by disrupting cytoskeletal organization. The process is mediated via the downstream component, aSpectrin, which localizes to the EnVs. Our data provide new insight into CLP function in tumors and identifies specific targets for tumor control.

Impact of Gene Repression on Biofilm Formation of Vibrio cholerae.

Vibrio cholerae, the etiological agent of cholera, is a facultative intestinal pathogen which can also survive in aquatic ecosystems in the form of biofilms, surface-associated microbial aggregates embedded in an extracellular matrix, which protects them from predators and hostile environmental factors. Biofilm-derived bacteria and biofilm aggregates are considered a likely source for cholera infections, underscoring the importance of V. cholerae biofilm research not just to better understand bacterial ecology, but also cholera pathogenesis in the human host. While several studies focused on factors induced during biofilm formation, genes repressed during this persistence stage have been fairly neglected. In order to complement these previous studies, we used a single cell-based transcriptional reporter system named TetR-controlled recombination-based in-biofilm expression technology (TRIBET) and identified 192 genes to be specifically repressed by V. cholerae during biofilm formation. Predicted functions of in-biofilm repressed (ibr) genes range from metabolism, regulation, surface association, transmembrane transport as well as motility and chemotaxis. Constitutive (over)-expression of these genes affected static and dynamic biofilm formation of V. cholerae at different stages. Notably, timed expression of one candidate in mature biofilms induced their rapid dispersal. Thus, genes repressed during biofilm formation are not only dispensable for this persistence stage, but their presence can interfere with ordered biofilm development. This work thus contributes new insights into gene silencing during biofilm formation of V. cholerae.

An Intranasal Vaccine Based on Outer Membrane Vesicles Against SARS-CoV-2.

The prevailing pandemic of SARS-CoV-2 highlights the desperate need of alternative vaccine-platforms, which are safe, effective, and can be modified to carry antigens of emerging pathogens. The current SARS-CoV-2 vaccines based on mRNA and adenoviral vector technology meet some of these criteria but still face limitations regarding administration route, mass production, stability, and storage. Herein, we introduce a novel SARS-CoV-2 vaccine candidate based on bacterial outer membrane vesicles (OMVs). Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) have been genetically modified to produce increased amounts of detoxified OMVs decorated with the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Intranasal immunization with RBD-decorated OMVs induced not only a robust immune response against the bacterial outer membrane components but also detectable antibody titers against the Spike protein. Cell culture infection assays using a Spike-pseudotyped lentivirus confirmed the presence of SARS-CoV-2 neutralizing antibodies. Highest titers against the SARS-CoV-2 Spike protein and most potent neutralization activity were observed for an alternating immunization regimen using RBD-decorated OMVs from ETEC and V. cholerae in turn. These results highlight the versatile vaccine applications offered by OMVs via expression of heterologous antigens in the donor bacterium.

Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake.

Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.