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Successful subversion of translation initiation factors 4E and 4G determines the infection success of potyviruses, the largest group of viruses affecting plants. In the natural variability of many plant species, resistance to potyvirus infection is provided by polymorphisms at4Eand4Gthat renders them inadequate for virus hijacking but still functional in translation initiation. In crops where such natural resistance alleles are limited, the genetic inactivation of4Ehas been proposed for the engineering of potyvirus resistance. However, recent findings indicate that knockout4Eand4Galleles may be deleterious for plant health and could jeopardize resistance efficiency in comparison to functional resistance proteins. Here, we explored the cause of these adverse effects by studying the role of theArabidopsis eIF4E1, whose inactivation was previously reported as conferring resistance to the potyvirus clover yellow vein virus (ClYVV) while also promoting susceptibility to another potyvirus called turnip mosaic virus (TuMV). We report thateIF4E1is required to maintain global plant translation and to restrict TuMV accumulation during infection, and its absence is associated with a favoured virus multiplication over host translation. Furthermore, our findings demonstrate that eIF4E1 plays a crucial role in inhibiting the TuMV-induced degradation of the translation initiation factor eIFiso4G1, thereby preventing the generation of a truncated protein. Finally, we demonstrate a role for eIFiso4G1 in TuMV accumulation and in supporting plant fitness during infection. These findings suggest that eIF4E1 counteracts the hijacking of the plant translational apparatus during TuMV infection and underscore the importance of preserving the functionality of translation initiation factors 4E and 4G when implementing potyvirus resistance strategies.Author summaryPlants are constantly under threat from viruses that can damage crops and reduce yield. Among these viruses, potyviruses are a major concern, and a small group of genes known aseIF4Eare key factors in making a plant susceptible to them. To combat these viruses, it is possible to either use naturally-selected variants ofeIF4Ethat provide resistance, or to disable the gene altogether. However, new research has shown that inactivatingeIF4Egenes may have unintended consequences for the plant’s development while compromise resistance to other potyviruses.To investigate this further, we focus in this work on the role of theArabidopsis eIF4E1whose inactivation confers resistance to one potyvirus, clover yellow vein virus (ClYVV). We looked why this same mutation ateIF4E1makes the plants more susceptible to another potyvirus, turnip mosaic virus (TuMV). Our study reveals that eIF4E1 acts in safeguarding the plant translational machinery during TuMV infection. By preventing the degradation of the translation initiation protein eIFiso4G1, eIF4E1 enables the plant to maintain its normal translation activity and ultimately prevents the accumulation of virus proteins.Our findings provide valuable insights into how potyviruses hijack the plant’s translation process, and emphasizes the need of preserving the functionality of translation initiation factors when developing potyvirus resistances.

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

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.

Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of theSMN1gene in most cases or mutations in rare cases. Interestingly, severalSMN1mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that inSMN1mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMRFM). Importantly, SMA-linked SMNTUDORmutant forms (SMNST) failed to interact with FMRFM. In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.

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.

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.

Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalysed by the terminal reductases and as endogenous electron shuttles. In the model organism Shewanella oneidensis, riboflavin production and excretion triggers a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescin decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations which by far exceed the necessary threshold value.ImportanceFerric iron is the fourth most abundant element of the earth crust. It occurs at neutral pH in the form insoluble iron minerals. The dissimilatory reduction of these minerals is an import part of global geological cycles and is catalyzed by microorganisms through extended respiratory chains to the cell surface. Shewanella oneidensis is one of the best understood model organisms for this kind of extracellular respiration. Flavins are important for the reduction of extracellular electron acceptors by S. oneidensis. since they have a function as (I) cofactors of the terminal reductases and (II) electron shuttles. In this study we reveal that flavin molecules are further employed as quorum sensing molecules. They are excreted by the organisms in a growth dependent manner and lead to anaerobic biofilm formation as a specific response at a certain threshold concentration. Although we know multiple examples of quorum sensing mechanisms, the use of riboflavin was so far not described and at least in S. oneidensis proceeds via a new regulatory routine that proceeds on the trancriptomic and posttranscriptomic level.

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.

International audience; 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 .

Pseudomonas aeruginosais 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 inP. aeruginosa.HighlightsT3SS substrates are secreted sequentially but information on the switches are missingInteraction of the T3SS ATPase with secreted proteins were investigated by different approachesMicroscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexesThe Gate-keeper complex binds to the ATPase and increases its affinity for the needle complexA new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase

Background Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. This process can be applied in bioelectrochemical systems in which the organisms produce an electrical current by respiring with an anode as electron acceptor. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalyzed by the terminal reductases and in some cases as endogenous electron shuttles. Results In the model organism Shewanella oneidensis , riboflavin production and excretion trigger a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum-sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescine decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations, which by far exceed the necessary threshold value. Conclusion This study describes a new quorum-sensing mechanism in S. oneidensis . Biofilm formation of S. oneidensis is induced by low concentrations of riboflavin resulting in an upregulation of the ornithine-decarboxylase speC . The results can be applied for the development of strains catalyzing increased current densities in bioelectrochemical systems.