SummaryAfter uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago± lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis. We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago±lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membranebound vacuole expressing the late endosomal marker, LAMP1, and the sec61b protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.

Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes – needed for invading epithelial cells – were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl . Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

A murine osmotic demyelination syndrome (ODS) model of the central nervous system included the relay thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei. Morphologic comparisons between treatments have revealed oligodendrocyte changes and, already 12 hours following the osmolality restoration, some heavily contrasted oligodendrocytes formed a unique intracellular primary cilium. This unique structure, found in vivo, in mature CNS oligodendrocytes, could account for a local awakening of some of the developmental proteome as it can be expressed in oligodendrocyte precursor cells. This resilience accompanied the emergence of arl13b protein expression along with restoration of nerve cell body axon hillocks shown in a previous issue of this journal. Additionally, the return of several thalamic oligodendrocyte fine features (nucleus, organelles) was shown 36 h later, including some mitosis. Those cell restorations and recognized translational activities comforted that local repairs could again take place, due to oligodendrocyte resilience after ODS instead or added to a postulated immigration of oligodendrocyte precursor cells distant from the sites of myelinolysis.