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Stressed bacteria can enter a dormant viable but non-culturable (VBNC) state. VBNC pathogens pose an increased health risk as they are undetectable by growth-based techniques and can wake up back into a virulent state. Although widespread in bacteria, the mechanisms governing this phenotypic switch remain elusive. Here, we investigate the VBNC state transition in the human pathogen Listeria monocytogenes. We show that bacteria starved in mineral water become VBNC by converting into osmotically stable cell wall-deficient coccoid forms, a phenomenon that occurs in other Listeria species. We reveal the bacterial stress response regulator SigB and the autolysin NamA as major actors of VBNC state transition. We lastly show that VBNC Listeria revert to a walled and virulent state after passage in chicken embryos. Our study provides more detail on the VBNC state transition mechanisms, revealing wall-free bacteria naturally arising in aquatic environments as a potential survival strategy in hypoosmotic and oligotrophic conditions., (© 2024. The Author(s).)

This protocol describes how to analyze C. albicans biofilm using transmission electron microscopy. We present two approaches to observe the ultrastructure of fungal cells within unperturbed biofilms, as well as an immunogold labeling procedure. This approach maintains the architecture of the fungal biofilm close to its native state by growing C. albicans biofilm on a plastic surface. After the freeze substitution procedure, classical transmission electron microscopy or electron tomography will allow the ultrastructural analysis of the microbial community., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Viruses of the giant virus family are characterized by a structurally conserved scaffold-capsid protein that shapes the icosahedral virion. The vaccinia virus (VACV) scaffold protein D13, however, transiently shapes the newly assembled viral membrane in to a sphere and is absent from the mature brick-shaped virion. In infected cells D13, a 62 kDa polypeptide, forms trimers that arrange in hexamers and a honey-comb like lattice. Membrane association of the D13-lattice may be mediated by A17, an abundant 21 kDa viral membrane protein. Whether membrane binding mediates the formation of the honey-comb lattice or if other factors are involved, remains elusive. Here we show that H7, a 17 kDa protein conserved among poxviruses, mediates proper formation of D13-hexamers, and hence the honey comb lattice and spherical immature virus. Without H7 synthesis D13 trimers assemble into a large 3D network rather than the typical well organized scaffold layer observed in wild-type infection, composed of short D13 tubes of discrete length that are tightly associated with the endoplasmic reticulum (ER). The data show an unexpected role for H7 in D13 organization and imply that formation of the honey-comb, hexagonal, lattice is essential for VACV membrane assembly and production of infectious progeny. The data are discussed with respect to scaffold proteins of other giant viruses., (© 2022. The Author(s).)

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenic mechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, loss of which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomic studies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels as putative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We also confirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disrupts mitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in our mouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues., Competing Interests: Disclosures: F. Darios reports "other" from Dynacure SAS outside the submitted work. D. Galatolo is supported by a grant from Treat SPG56. G. Stevanin reports grants from ASL-HSP association, the Tom-Wahlig-Stiftung Foundation, and the European Union 7th Framework Programme (Neuromics); non-financial support from Agence Nationale de la Recherche (framework programme Investissements d'Avenir) during the conduct of the study; and grants from Biogen outside the submitted work. N. Vilain reports grants from Fondation Bettencourt-Schueller, Fondation Servier, Union Nationale pour les Intérêts de la Médecine, and Fondation pour la Recherche sur l'Alzheimer; non-financial support from Movement Disorders Society, Merz-Pharma, and GE Healthcare SAS; and "other" from Biogen, Eisai, Eli-Lilly, Roche, Janssen - Johnson & Johnson, and Alector outside the submitted work. No other disclosures were reported., (© 2021 Pujol et al.)