Search

Most bacteria are protected from environmental offenses by a cell wall consisting of strong yet elastic peptidoglycan. The cell wall is essential for preserving bacterial morphology and viability, and thus the enzymes involved in the production and turnover of peptidoglycan have become preferred targets for many of our most successful antibiotics. In the past decades, Vibrio cholerae, the gram-negative pathogen causing the diarrheal disease cholera, has become a major model for understanding cell wall genetics, biochemistry, and physiology. More than 100 articles have shed light on novel cell wall genetic determinants, regulatory links, and adaptive mechanisms. Here we provide the first comprehensive review of V. cholerae’s cell wall biology and genetics. Special emphasis is placed on the similarities and differences with Escherichia coli, the paradigm for understanding cell wall metabolism and chemical structure in gram-negative bacteria.

Peptidoglycan (PG) has remained for decades in the spotlight of the never-ending battle against pathogenic bacteria as this essential bacterial structure is one of the most successful targets for antibiotics. Most of our current understanding about the composition, architecture, and dynamics of the PG relies on techniques which have experienced great technological and methodological improvements in the past years. Here we summarize recent advances in these methods with the intention to furnish a valuable resource for both PG experts and newcomers.

Peripheral nerve section with subsequent disconnection of motor neuron (MN) cell bodies from their skeletal muscle targets leads to a rapid reactive response involving the recruitment and activation of microglia. In addition, the loss of afferent synapses on MNs occurs in concomitance with microglial reaction by a process described as synaptic stripping. However, the way in which postaxotomy‐activated microglia adjacent to MNs are involved in synaptic removal is less defined. Here, we used confocal and electron microscopy to examine interactions between recruited microglial cells and presynaptic terminals in axotomized MNs between 1 and 15 days after sciatic nerve transection in mice. We did not observe any bulk engulfment of synaptic boutons by microglia. Instead, microglial cells internalized small membranous‐vesicular fragments which originated from the acute disruption of synaptic terminals involving the activation of the necroptotic pathway. The presence of abundant extracellular vesicles in the perineuronal space after axotomy, together with the increased expression of phospho‐mixed lineage kinase domain‐like protein and, later, of extracellular vesicle markers, such as CD9, CD63, and flotillin, indicate that the vesicles mainly originated in synapses and were transferred to microglia. The upregulation of Rab7 and Rab10 in microglia interacting with injured MNs, indicated the activation of endocytosis. As activated microglia and synaptic boutons displayed positive C1q immunoreactivity, a complement‐mediated opsonization may also contribute to microglial‐mediated synaptic disruption. In addition to the relevance of our data in the context of neuroinflammation and MN disease, they should also be taken into account for understanding functional recovery after peripheral nerve injury., Main Points Early after axotomy, microglia recruited near injured motor neurons, emit processes that tend to contact their afferent synaptic terminals.Extracellular vesicles resulting from necroptotic synaptic disruption are removed by microglia.

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold but persists in cold waters in the form of viable but nondividing coccoidal-shaped cells. Additionally, V. cholerae is able to form nonproliferating spherical cells in response to cell wall damage. It was recently reported that l-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that l-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable, and upon removal of l-arabinose, they start expanding in volume, form branched structures, and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of high-level expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of l-arabinose by the V. cholerae homolog of the Escherichia coli galactose transport and catabolic system. Taken together, our results suggest that the formation of nongrowing spherical cells is a common response of vibrios exposed to detrimental conditions. They also permit us to define conditions preventing any physiological perturbation of V. cholerae when using l-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coliaraBAD operon. IMPORTANCEVibrio spp., among other bacteria, form transient cell wall-deficient forms as a response to different stresses and revert to proliferating rods when permissive conditions have been restored. Such cellular forms have been associated with antimicrobial tolerance, chronic infections, and environmental dispersion. The effect of l-Ara on V. cholerae could provide an easily tractable model to study the ability of vibrios to form viable reversible spheroplasts. Indeed, the quick transition to spheroplasts and reversion to proliferating rods by addition or removal of l-Ara are ideal to understand the genetic program governing this physiological state and the spatial rearrangements of the cellular machineries during cell shape transitions.

14 páginas.- 6 figuras.- 2 tablas.- referencias.- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.647305/full#supplementary-material, Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA− and ZapB− mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile, This study was supported by grant BIO2016-75235-P from the Spanish Ministerio de Economía, Industria y Competitividad - Agencia Estatal de Investigación and the European Regional Fund. VU was supported by a postdoctoral contract associated to grant CVI-5879 from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain. MS-R was supported by the VI Plan Propio de Investigación y Transferencia de la Universidad de Sevilla.

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold but persists in cold waters in the form of viable but nondividing coccoidal-shaped cells. Additionally, V. cholerae is able to form nonproliferating spherical cells in response to cell wall damage. It was recently reported that l-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that l-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable, and upon removal of l-arabinose, they start expanding in volume, form branched structures, and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of high-level expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of l-arabinose by the V. cholerae homolog of the Escherichia coli galactose transport and catabolic system. Taken together, our results suggest that the formation of nongrowing spherical cells is a common response of vibrios exposed to detrimental conditions. They also permit us to define conditions preventing any physiological perturbation of V. cholerae when using l-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coli araBAD operon. IMPORTANCE Vibrio spp., among other bacteria, form transient cell wall-deficient forms as a response to different stresses and revert to proliferating rods when permissive conditions have been restored. Such cellular forms have been associated with antimicrobial tolerance, chronic infections, and environmental dispersion. The effect of l-Ara on V. cholerae could provide an easily tractable model to study the ability of vibrios to form viable reversible spheroplasts. Indeed, the quick transition to spheroplasts and reversion to proliferating rods by addition or removal of l-Ara are ideal to understand the genetic program governing this physiological state and the spatial rearrangements of the cellular machineries during cell shape transitions.

A general survival strategy of many life forms faced with harmful growth conditions is to enter into a non-proliferating state until conditions suitable for growth are restored. In bacteria, this survival strategy is associated with antimicrobial tolerance, chronic infections and environmental dispersion. In particular, the agent of the deadly human disease cholera,Vibrio cholerae, undergoes a morphological transition from a rod-shaped proliferative form to a spherical non-proliferating form after exposure to cold or cell wall targeting antibiotics. Growth is resumed when the adverse conditions have ceased.Here, we show that a component of the hemicellulose and pectin of terrestrial plants, L-arabinose, triggers the formation of non-proliferatingV. choleraespherical cells, which are able to return to growth when L-arabinose is removed from the growth medium. We found that the cell wall of L-arabinose treatedV. choleraecells has a peptidoglycan composition similar to the cell wall of spheroplasts and that they revert to a wild-type morphology through the formation of branched cells like L-forms. Unlike L-forms, however, they are osmo-resistant. Through a random Tn genetic screen for mutants insensitive to L-arabinose, we identified genes involved in the uptake and catabolism of galactose and in glycolysis. We hypothesize that L-arabinose is enzymatically processed by the galactose catabolism and glycolysis pathways until it is transformed in a product that cannot be further recognized byV. choleraeenzymes. Accumulation of this enzymatic by-product triggers the formation of viable non-dividing cell wall deficient spherical cells.

International audience; Enterococcus faecium has become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most prescribed xenobiotics for ICU patients on fitness, pathogenicity and antimicrobial resistance of the vanB-positive E. faecium Aus0004 reference strain. Several phenotypic analyses were carried out and we observed that caspofungin, an antifungal agent belonging to the echinocandins family, had an important effect on E. faecium growth in vitro. We confirmed this effect by electron microscopy and peptidoglycan analysis and showed that, even at a subinhibitory concentration (¼ × MIC, 8 mg/L), caspofungin had an impact on cell wall organization especially in abundance of some muropeptide precursors. By RNA-seq, it was also shown that around 20% of the transcriptome were altered in the presence of caspofungin with 321 and 259 significantly upregulated and downregulated genes, respectively. Since the fungal target of caspofungin (i.e., beta-(1,3)-glucan synthase) was absent in bacteria, the mechanistic pathway of caspofungin activity was investigated. The repression of genes involved in the pyruvate metabolism seemed to have a drastic impact on bacterial cell viability while decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan. This is the first report of caspofungin antibacterial activity against E. faecium, highlighting the potential impact of non-antibiotic xenobiotics against bacterial pathogens.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both. Agence Nationale de la Recherche ANR-15-CE11-0001-02 Centre National de la Recherche Scientifique PICS07279 French State Program "Investissements d'Avenir" ANR-11-LABX-0011

Summary: The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for the maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized and recycled. Here, we show that Vibrio cholerae recycles PG fragments modified with non-canonical d-amino acids (NCDAA), which lead to the accumulation of cytosolic PG tetrapeptides. We demonstrate that the accumulation of recycled tetrapeptides has two regulatory consequences for the cell wall: reduction of d,d-cross-linkage and reduction of PG synthesis. We further demonstrate that l,d-carboxypeptidases from five different species show a preferential activity for substrates containing canonical (d-alanine) versus non-canonical (d-methionine) d-amino acids, suggesting that the accumulation of intracellular tetrapeptides in NCDAA-rich environments is widespread. Collectively, this work reveals a regulatory role of NCDAA linking PG recycling and synthesis to promote optimal cell wall assembly and composition in the stationary phase. : A critical step in peptidoglycan (PG) recycling is the transformation of PG tetrapeptides into tripeptides. Hernández et al. demonstrate that Vibrio cholerae accumulates tetrapeptide PG precursors to downregulate PG synthesis in the stationary phase. Tetrapeptide accumulation relies on the substrate preference of l,d-carboxypeptidases for d-ala versus NCDAA-modified substrates. Keywords: cell wall, Vibrio cholerae, peptidoglycan recycling, l,d-carboxypeptidase, l,d-transpeptidase, NCDAA, d-amino acids, Tn-seq

The hyperproduction of the chromosomal AmpC beta-lactamase is the main mechanism driving beta-lactam resistance in Pseudomonas aeruginosa, one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress ampC expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent P. aeruginosa stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient ampC hyperexpression during cefoxitin induction (1,6-anhydro-N-acetylmuramyl-pentapeptides) also underlie the dacB (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro-N-acetylmuramyl-tripeptides notably overaccumulated in the ampD knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro-Nacetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of beta-lactamase production, dominate over the repressor (UDP-N-acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent beta-lactam resistance in P. aeruginosa, potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling. IMPORTANCE The extensive use of beta-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa, the mutation-driven AmpC beta-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern ampC hyperexpression and resistance., Work in the Oliver lab is supported by the Sociedad Espanola de Enfermedades Infecciosas y Microbiologia Clinica (SEIMC), the Ministerio de Economia y Competitividad of Spain, and the Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF; A way to achieve Europe) through the Spanish Network for the Research in Infectious Diseases (RD12/0015 and RD16/0016), and grants CP12/03324, PI15/00088, PI15/02212, PI18/00681, and PI18/00076. Work in the Cava lab is supported by the Swedish Research Council (VR), the Knut and Alice Wallenberg Foundation (KAW), the Laboratory of Molecular Infection Medicine Sweden (MIMS), and the Kempe Foundation.

Enzymes catalysing the stereo-chemical inter-conversion of amino acids are known as amino acid racemases. In bacteria, these enzymes are fundamental to synthesize the D-Ala and D-Glu that are critical components of the peptidoglycan. In addition to this structural function in cell wall assembly, D-amino acids produced by microbial amino acid racemases have been described as relevant constituents in other prokaryotic structures (e.g. capsule, non-ribosomal peptides) and have been associated to growth fitness and to processes such as biofilm development, spore germination and signalling. The recent discovery of broad spectrum racemases able to produce and release several D-amino acids to the environment suggests that these enzymes might have a great impact in microbial ecology. Consequently, new data on the biochemistry and regulation of racemases is key to understand the biological significance of D-enantiomers in nature, in particular their effect on microbial social networks. This review summarizes current knowledge on the environmental roles of bacterial racemases with an emphasis on the potential roles of the new broad spectrum enzymes in natural environments.

Changes in the peptidoglycan (PG) structure of Salmonella enterica are detected in the presence of a sublethal concentration of sodium deoxycholate (DOC): (i) lower proportions of Braun lipoprotein ...

In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila , outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d , d -carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila . In a heterologous assembly model in E. coli , expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.

High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms ...

Enzymes catalysing the stereo-chemical inter-conversion of amino acids are known as amino acid racemases. In bacteria, these enzymes are fundamental to synthesize the D-Ala and D-Glu that are critical components of the peptidoglycan. In addition to this structural function in cell wall assembly, D-amino acids produced by microbial amino acid racemases have been described as relevant constituents in other prokaryotic structures (e.g. capsule, non-ribosomal peptides) and have been associated to growth fitness and to processes such as biofilm development, spore germination and signalling. The recent discovery of broad spectrum racemases able to produce and release several D-amino acids to the environment suggests that these enzymes might have a great impact in microbial ecology. Consequently, new data on the biochemistry and regulation of racemases is key to understand the biological significance of D-enantiomers in nature, in particular their effect on microbial social networks. This review summarizes current knowledge on the environmental roles of bacterial racemases with an emphasis on the potential roles of the new broad spectrum enzymes in natural environments.

The antibacterial activities of bile salts include detergent activity on cell membranes, protein denaturation, and DNA damage. However, many bacterial species are resistant to the antibacterial activity of bile salts. Bile salts may thus be viewed both as antibacterial compounds and as signals used by bacteria to identify bile-containing animal environments. This chapter reviews the mechanisms employed by Escherichia coli, Salmonella enterica, and other intestinal bacteria to respond to bile salts, with emphasis on their relevance for pathogenesis. The reader is also referred to two comprehensive, insightful reviews on bacterial responses to bile. Unconjugated forms of bile salts are amphipatic molecules that can enter the bacterial cell by crossing membrane bilayers. Furthermore, in the outer membrane of gram-negative bacteria, porins provide passage to both unconjugated and conjugated bile salts. Bile salts, especially if unconjugated, may easily reach the bacterial ctyoplasm by diffusion. Bile salts may also bind to specific receptors in the bacterial surface, activating signal transduction pathways that change gene expression patterns. Bile extract or individual bile salts can be added to microbiological media, thus permitting reductionist studies of bacterial bile resistance in the laboratory. It is hypothesized that nonmutational, reversible resistance may involve stochastic activation of one or more efflux systems that transport bile outside the cell.

Changes in the peptidoglycan (PG) structure of Salmonella enterica are detected in the presence of a sublethal concentration of sodium deoxycholate (DOC): (i) lower proportions of Braun lipoprotein (Lpp)-bound muropeptides; (ii) reduced levels of muropeptides cross-linked by L(meso)-diaminopimelyl-D(meso)-diaminopimelic acid (L-D) peptide bridges (3-3 cross-links). Similar structural changes are found in S. enterica cultures adapted to grow in the presence of a lethal concentration of DOC, suggesting that reduced anchoring of Braun protein to PG and low occurrence of 3-3 cross-links may increase S. enterica resistance to bile. This view is further supported by additional observations: (i) A triple mutant lacking L,D-transpeptidases YbiS, ErfK, and YcfS, which does not contain Lpp anchored to PG, is hyper-resistant to bile; (ii) enhanced 3-3 cross-linking upon overexpression of YnhG transpeptidase causes a decrease in bile resistance. These observations suggest that remodelling of the cell wall may be added to the list of adaptive responses that permit survival of S. enterica in the presence of bile.

Prc is a periplasmic protease involved in processing of penicillin-binding protein 3 (PBP3). Lack of Prc suppresses bile sensitivity in Dam-, Wec-, PhoP-, DamX-, and SeqA- mutants of Salmonella enterica, and increases bile resistance in the wild type. Changes in the activity of penicillin binding proteins PBP3, PBP4, PBP5/6 and PBP7 are detected in a Prc- background, suggesting that peptidoglycan remodeling might contribute to bile resistance.

Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization. ISSN:1932-6203

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease with no cure. Currently there are only two ALS drugs approved by the FDA, both with a limited therapeutic effect. In the search for drug candidates for ALS, we studied the effect of known stem cell mobilizing agents (treatment) and antimetabolite 5-fluorouracil (5-FU) (anti-treatment) in SOD1G93A model of ALS. Surprisingly, we found that anti-cancer drug 5-FU increases lifespan, delays the disease onset and improves motor performance in ALS mice. Although we were not able to demonstrate the mechanistic basis of the beneficial 5-FU action in ALS mice, our findings suggest that 5-FU or similar drugs are possible drug candidates for the treatment of motor neuron diseases through drug repurposing. This work was funded by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union (Grants PI14/00947 and PI17/00949), Asociación Adelante de La Roda, Plataforma Afectados por la ELA, Asociación Juntos Venceremos ELA, AVPA Zaragoza (RO), TERCEL (RD12/0019/0011 and RD16/0011/0035) and CIBERNED (CB06/05/1105) funds from the Instituto de Salud Carlos III of Spain (XN), and Fondazione Roma and Agenzia Spaziale Italiana (AM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The electric activity of lower motor neurons (MNs) appears to play a role in determining cell-vulnerability in MN diseases. MN excitability is modulated by cholinergic inputs through C-type synaptic boutons, which display an endoplasmic reticulum-related subsurface cistern (SSC) adjacent to the postsynaptic membrane. Besides cholinergic molecules, a constellation of proteins involved in different signal-transduction pathways are clustered at C-type synaptic sites (M2 muscarinic receptors, Kv2.1 potassium channels, Ca2+ activated K+ [SK] channels, and sigma-1 receptors [S1R]), but their collective functional significance so far remains unknown. We have previously suggested that neuregulin-1 (NRG1)/ErbBs-based retrograde signalling occurs at this synapse. To better understand signalling through C-boutons, we performed an analysis of the distribution of C-bouton-associated signalling proteins. We show that within SSC, S1R, Kv2.1 and NRG1 are clustered in highly specific, non-overlapping, microdomains, whereas ErbB2 and ErbB4 are present in the adjacent presynaptic compartment. This organization may define highly ordered and spatially restricted sites for different signal-transduction pathways. SSC associated proteins are disrupted in axotomised MNs together with the activation of microglia, which display a positive chemotactism to C-bouton sites. This indicates that C-bouton associated molecules are also involved in neuroinflammatory signalling in diseased MNs, emerging as new potential therapeutic targets. We would like to thank Dr. Ronald W. Oppenheim for his critical reading of the manuscript and for helpful comments and suggestions. We would also like to thank Marta Hereu for her technical assistance, Lidia Delgado, Gema Marta Martínez and M. Yolanda Muela, from the Unitat de Criomicroscòpia Electrònica (Centres Científics i Tecnològics de la Universitat de Barcelona), for their technical support with ultrastructural immunolabelling, Daniel Cabezas for his help in some experiments, and the SCT animal facility of the Universitat de Lleida for mouse care and housing. This work was supported by grants from the Ministerio de Economía y Competitividad cofinanced by FEDER (SAF2015–70801-R to J.E.E. and J.C.), and from Jack Van den Hoek a la investigació de l’ELA - Fundació Miquel Valls (to J.E.E.).