Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases

Emergence and Modular Evolution of a Novel Motility Machinery in Bacteria

Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena

A novel class of bacterial motors involved in the directional transport of a sugar at the bacterial surface : The machineries of motility and sporulation in Myxococcus xanthus.

Le mécanisme de la motilité de type gliding chez Myxococcus xanthus est longtemps resté incompris, du fait que ce type de déplacement ne requière aucune organelle extracellulaire. Nous avons démontré que le gliding est énergisée par un canal à protons, composé par les protéines AglRQS. Ce moteur coopère avec le cytosquelette d’actine bactérien pour transporter de manière directionnelle le complexe de l’enveloppe Glt à la surface de la cellule. Ce transport est traduit en motilité car les complexes Glt transportés interagissent avec un polysaccharide de surface qui agit comme une colle et immobilise les complexes Glt transportés contre le substrat.Nous avons également fait l’étonnante découverte que le moteur AglRQS est également essentiel à la sporulation, processus cellulaire durant lequel les cellules s’arrondissent et sont recouvertes d’un épais polysaccharide (le spore coat), qui leur confère une résistance face à des conditions défavorables. Nous avons démontré une interaction directe entre le moteur AglRQS et le complexe de l’enveloppe Nfs, un proche homologue du complexe Glt. Nous avons démontré que le moteur AglRQS transporte le complexe Nfs de manière directionnelle autour de la spore. Le spore coat étant sécrété en différents foci autour de la surface de la spore, son transport par la machinerie Agl-Nfs assure la formation d’une couche de « spore coat » compacte autour de la future spore.Ces résultats démontrent l’existence d’un moteur bactérien impliqué dans le transport directionnel de complexes protéiques associés à des sucres. Ces moteurs modulaires pourraient être adaptés à des fonctions spécifiques, en fonction du complexe avec lequel ils interagissent.How gliding motility on solid surfaces is achieved in Myxococcus xanthus has long remained enigmatic, mostly because movement does not involve obvious extracellular organelles. Recently, we demonstrated that motility in M. xanthus is driven by a proton channel composed by the AglRQS proteins. This motor cooperates with the bacterial actin cytoskeleton to transport an envelope-spanning Glt motility complexes at the cell surface directionally. Motility is produced as a motility machinery surface tip-bound polysaccharide acts like a glue to immobilize the transported Glt complexes against the substratum.In the course of this study, we also made the surprising discovery that the AglRQS motor is essential not only for motility but also for sporulation, a cellular process during which the cells become surrounded by a thick polysaccharide (the spore coat) that confers resistance during unfavourable conditions. We demonstrated a direct interaction between the AglRQS motor and the Nfs envelope complex, a close homolog of the Glt complex. Transmission electron microscopy, time-lapse microscopy and localization studies, showed that the AglRQS motor rotates the Nfs complex directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by the Agl-Nfs machinery ensures the formation of a compact spore coat layer around the future spore.These results highlight the existence of new class of bacterial motors involved in intracellular and directional transport of sugar-associated complex. These modular motors can be adapted to specific functions based which output complex they interact with

Human Brain Organoids as Models for Central Nervous System Viral Infection

Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier

Functional Intercellular Transmission of miHTT via Extracellular Vesicles: An In Vitro Proof-of-Mechanism Study

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by GAG expansion in exon 1 of the huntingtin (HTT) gene. AAV5-miHTT is an adeno-associated virus serotype 5-based vector expressing an engineered HTT-targeting microRNA (miHTT). Preclinical studies demonstrate the brain-wide spread of AAV5-miHTT following a single intrastriatal injection, which is partly mediated by neuronal transport. miHTT has been previously associated with extracellular vesicles (EVs), but whether EVs mediate the intercellular transmission of miHTT remains unknown. A contactless culture system was used to evaluate the transport of miHTT, either from a donor cell line overexpressing miHTT or AAV5-miHTT transduced neurons. Transfer of miHTT to recipient (HEK-293T, HeLa, and HD patient-derived neurons) cells was observed, which significantly reduced HTT mRNA levels. miHTT was present in EV-enriched fractions isolated from culture media. Immunocytochemical and in situ hybridization experiments showed that the signal for miHTT and EV markers co-localized, confirming the transport of miHTT within EVs. In summary, we provide evidence that an engineered miRNA—miHTT—is loaded into EVs, transported across extracellular space, and taken up by neighboring cells, and importantly, that miHTT is active in recipient cells downregulating HTT expression. This represents an additional mechanism contributing to the widespread biodistribution of AAV5-miHTT

Phenotype inference in an Escherichia coli strain panel

Understanding how genetic variation contributes to phenotypic differences is a fundamental question in biology. Combining high-throughput gene function assays with mechanistic models of the impact of genetic variants is a promising alternative to genome-wide association studies. Here we have assembled a large panel of 696 Escherichia coli strains, which we have genotyped and measured their phenotypic profile across 214 growth conditions. We integrated variant effect predictors to derive gene-level probabilities of loss of function for every gene across all strains. Finally, we combined these probabilities with information on conditional gene essentiality in the reference K-12 strain to compute the growth defects of each strain. Not only could we reliably predict these defects in up to 38% of tested conditions, but we could also directly identify the causal variants that were validated through complementation assays. Our work demonstrates the power of forward predictive models and the possibility of precision genetic interventions.ISSN:2050-084

A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in <i>Myxococcus xanthus</i>

<div><p>Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the <i>Myxococcus xanthus</i> Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.</p></div

The Agl interacts with Nfs to promote spore coat assembly.

<p>(A) Sporulation titers after heat and sonication counted by DAPI staining in various strains. Corresponding DAPI-staining images are shown. Note the 10² spores/ml detection limit of the assay. Scale bar = 5 µm. (B) Thin sections of myxospores observed by transmission electron microscopy. WT, <i>aglQ</i>, <i>nfsD</i>, and <i>exoA</i> strains were observed 24 h after the induction of sporulation. Arrows point to spore coat material that detaches from the surface of sporulating cells in <i>aglQ</i> and <i>nfsD</i> mutants. Scale bars = 0.1 µm. (C) GSLI-FITC staining of the spore coat material. WT, <i>aglQ</i>, <i>nfsD</i>, and <i>exoA</i> strains were observed 4 h after the induction of sporulation. Scale bar = 1 µm. (D) AglR interacts with GltG and NfsG in a bacterial two hybrid assay.</p