A Micrarchaeon Isolate Is Covered by a Proteinaceous S-Layer.

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in "Ca. Micrarchaeum harzensis A_DKE," and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated, and the micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described coculture, containing "Ca. Micrarchaeum harzensis A_DKE" and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of "Ca. Micrarchaeum harzensis A_DKE." Both organisms are clearly distinguishable by cell size, shape, and surface structure. IMPORTANCE Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member "Candidatus Micrarchaeum harzensis A_DKE," an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of "Ca. Micrarchaeum harzensis A_DKE" is an important step toward understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

Microglia modulate gliotransmission through the regulation of VAMP2 proteins in astrocytes.

Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

Membrane vesicles released by Lacticaseibacillus casei BL23 inhibit the biofilm formation of Salmonella Enteritidis.

Biofilms represent a major concern in the food industry and healthcare. The use of probiotic bacteria and their derivatives as an alternative to conventional treatments to fight biofilm development is a promising option that has provided convincing results in the last decades. Recently, membrane vesicles (MVs) produced by probiotics have generated considerable interest due to the diversity of roles they have been associated with. However, the antimicrobial activity of probiotic MVs remains to be studied. In this work, we showed that membrane vesicles produced by Lacticaseibacillus casei BL23 (LC-MVs) exhibited strong antibiofilm activity against Salmonella enterica serovar Enteritidis (S. Enteritidis) without affecting bacterial growth. Furthermore, we found that LC-MVs affected the early stages of S. Enteritidis biofilm development and prevented attachment of bacteria to polystyrene surfaces. Importantly, LC-MVs did not impact the biomass of already established biofilms. We also demonstrated that the antibiofilm activity depended on the proteins associated with the LC-MV fraction. Finally, two peptidoglycan hydrolases (PGHs) were found to be associated with the antibiofilm activity of LC-MVs. Overall, this work allowed to identify the antibiofilm properties of LC-MVs and paved the way for the use of probiotic MVs against the development of negative biofilms.

Spontaneous Prophage Induction Contributes to the Production of Membrane Vesicles by the Gram-Positive Bacterium Lacticaseibacillus casei BL23.

The formation of membrane vesicles (MVs) by Gram-positive bacteria has gained increasing attention over the last decade. Recently, models of vesicle formation have been proposed and involve the digestion of the cell wall by prophage-encoded or stress-induced peptidoglycan (PG) hydrolases and the inhibition of PG synthesis by ß-lactam antibiotics. The impact of these mechanisms on vesicle formation is largely dependent on the strain and growth conditions. To date, no information on the production of vesicles by the lactobacilli family has been reported. Here, we aimed to characterize the MVs released by the Gram-positive bacteria Lacticaseibacillus casei BL23 and also investigated the mechanisms involved in vesicle formation. Using electron microscopy, we established that the size of the majority of L. casei BL23 vesicles ranged from 50 to 100 nm. Furthermore, we showed that the vesicles were released consistently throughout the growth of the bacteria in standard culture conditions. The protein composition of the vesicles released in the supernatant was identified and a significant number of prophage proteins was detected. Moreover, using a mutant strain harboring a defective PLE2 prophage, we were able to show that the spontaneous and mitomycin-triggered induction of the prophage PLE2 contribute to the production of MVs by L. casei BL23. Finally, we also demonstrated the influence of prophages on the membrane integrity of bacteria. Overall, our results suggest a key role of the prophage PLE2 in the production of MVs by L. casei BL23 in the absence or presence of genotoxic stress. IMPORTANCE The last few decades have demonstrated that membrane vesicles (MVs) produced by microorganisms can have a wide variety of functions. This diversity places MVs at the crossroads of major research topics in current microbiology such as antibiotic resistance, horizontal gene transfer, cell communication, biofilm development, bacteriophage resistance, and pathogenesis. In particular, vesicles produced by probiotic strains have been shown to play a significant role in their beneficial effects. Thus, the study of vesicle biogenesis is a key element for promoting and improving their release. Overall, our results suggest a key role of spontaneous and mitomycin-triggered prophage induction in MV production by the Gram-positive bacteria Lacticaseibacillus casei BL23. This phenomenon is of great interest as prophage-induced MVs could potentially influence bacterial behavior, stress resistance, and vesicle functions.

Environmental cues from neural crest derivatives act as metastatic triggers in an embryonic neuroblastoma model.

Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers.

Developing Rhodobacter sphaeroides for cathodic biopolymer production.

In this work, Rhodobacter sphaeroides was identified as a potential cathodic production strain for photoautotrophic production processes. First, a stable cultivation in a bioelectrochemical system (BES) was established under conditions in which hydrogen produced by a poised cathode served as an electron donor. It was shown that both the introduction of a plasmid vector and exposure to the corresponding antibiotic selection pressure caused a strong improvement in both cathodic biofilm formation and electrochemical properties. A quantitative proteomic analysis identified key players in the molecular adaptation to biofilm growth on the cathodic surface. Furthermore, biofilm formation kinetics were quantified by optical coherence tomography measurements, which showed a strong tendency for biofilm formation together with a robust biofilm architecture. A media switch to N2-limited conditions resulted in increased cathodic poly(3-hydroxybutyrate) (PHB) accumulation, suggesting R. sphaeroides as a potential strain for photoautotrophic PHB production in future industrial applications.

Production of acetoin from renewable resources under heterotrophic and mixotrophic conditions.

This study aimed to reveal whether Cupriavidus necator H16 is suited for the production of acetoin based on the carboxylic acids acetate, butyrate and propionate under heterotrophic and mixotrophic conditions. The chosen production strain, lacking the polyhydroxybutyrate synthases phaC1 and phaC2, was revealed to be beneficiary for autotrophic acetoin production. Proteomic analysis of the strain determined that the deletions do indeed have a significant impact on pyruvate formation and its subsequent direction towards the introduced acetoin-synthesis pathway. Moreover, the strain was tested for its ability to use typical dark fermentation products under hetero- and mixotrophic conditions. Growth with butyrate and acetate led to low efficiencies, while 46.54% ±0.78 of the added propionate was converted into acetoin. Interestingly, mixotrophic conditions led to simultaneous consumption of acetate and butyrate with the gaseous substrates and lowered efficiency. In contrast, mixotrophic propionate consumption led to diauxic behavior and high carbon efficiency of 71.2% ±0.64.

The PopN Gate-keeper Complex Acts on the ATPase PscN to Regulate the T3SS Secretion Switch from Early to Middle Substrates in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa.