The expression of virulence genes increases membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium.

Virulence gene expression can represent a substantial fitness cost to pathogenic bacteria. In the model entero-pathogen Salmonella Typhimurium (S.Tm), such cost favors emergence of attenuated variants during infections that harbor mutations in transcriptional activators of virulence genes (e.g., hilD and hilC). Therefore, understanding the cost of virulence and how it relates to virulence regulation could allow the identification and modulation of ecological factors to drive the evolution of S.Tm toward attenuation. In this study, investigations of membrane status and stress resistance demonstrate that the wild-type (WT) expression level of virulence factors embedded in the envelope increases membrane permeability and sensitizes S.Tm to membrane stress. This is independent from a previously described growth defect associated with virulence gene expression in S.Tm. Pretreating the bacteria with sublethal stress inhibited virulence expression and increased stress resistance. This trade-off between virulence and stress resistance could explain the repression of virulence expression in response to harsh environments in S.Tm. Moreover, we show that virulence-associated stress sensitivity is a burden during infection in mice, contributing to the inherent instability of S.Tm virulence. As most bacterial pathogens critically rely on deploying virulence factors in their membrane, our findings could have a broad impact toward the development of antivirulence strategies.

Novel biochemical, structural, and systems insights into inflammatory signaling revealed by contextual interaction proteomics.

Protein-protein interactions (PPIs) represent the main mode of the proteome organization in the cell. In the last decade, several large-scale representations of PPI networks have captured generic aspects of the functional organization of network components but mostly lack the context of cellular states. However, the generation of context-dependent PPI networks is essential for structural and systems-level modeling of biological processes-a goal that remains an unsolved challenge. Here we describe an experimental/computational strategy to achieve a modeling of PPIs that considers contextual information. This strategy defines the composition, stoichiometry, temporal organization, and cellular requirements for the formation of target assemblies. We used this approach to generate an integrated model of the formation principles and architecture of a large signalosome, the TNF-receptor signaling complex (TNF-RSC). Overall, we show that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function.

Microbial genome collection of aerobic granular sludge cultivated in sequencing batch reactors using different carbon source mixtures.

Aerobic granular sludge (AGS) consists of a microbial consortium that has an important role in wastewater treatment. This study investigates AGS microorganisms cultivated in a laboratory-scale sequencing batch reactor. Metagenomic sequencing was conducted using PacBio and Illumina, resulting in 759 metagenome-assembled genomes, 331 of which remained after dereplication.

A novel EPM2A mutation yields a slow progression form of Lafora disease.

Lafora disease (LD, OMIM 254780) is a rare disorder characterized by epilepsy and neurodegeneration leading patients to a vegetative state and death, usually within the first decade from the onset of the first symptoms. In the vast majority of cases LD is related to mutations in either the EPM2A gene (encoding the glucan phosphatase laforin) or the EPM2B gene (encoding the E3-ubiquitin ligase malin). In this work, we characterize the mutations present in the EPM2A gene in a patient displaying a slow progression form of the disease. The patient is compound heterozygous with Y112X and N163D mutations in the corresponding alleles. In primary fibroblasts obtained from the patient, we analyzed the expression of the mutated alleles by quantitative real time PCR and found slightly lower levels of expression of the EPM2A gene respect to control cells. However, by Western blotting we were unable to detect endogenous levels of the protein in crude extracts from patient fibroblasts. The Y112X mutation would render a truncated protein lacking the phosphatase domain and likely degraded. Since minute amounts of laforin-N163D might still play a role in cell physiology, we analyzed the biochemical characteristics of the N163D mutation. We found that recombinant laforin N163D protein was as stable as wild type and exhibited near wild type phosphatase activity towards biologically relevant substrates. On the contrary, it showed a severe impairment in the interaction profile with previously identified laforin binding partners. These results lead us to conclude that the slow progression of the disease present in this patient could be either due to the specific biochemical properties of laforin N163D or to the presence of alternative genetic modifying factors separate from pathogenicity.