Molecular stripping underpins derepression of a toxin-antitoxin system.

Transcription factors control gene expression; among these, transcriptional repressors must liberate the promoter for derepression to occur. Toxin-antitoxin (TA) modules are bacterial elements that autoregulate their transcription by binding the promoter in a T:A ratio-dependent manner, known as conditional cooperativity. The molecular basis of how excess toxin triggers derepression has remained elusive, largely because monitoring the rearrangement of promoter-repressor complexes, which underpin derepression, is challenging. Here, we dissect the autoregulation of the Salmonella enterica tacAT3 module. Using a combination of assays targeting DNA binding and promoter activity, as well as structural characterization, we determine the essential TA and DNA elements required to control transcription, and we reconstitute a repression-to-derepression path. We demonstrate that excess toxin triggers molecular stripping of the repressor complex off the DNA through multiple allosteric changes causing DNA distortion and ultimately leading to derepression. Thus, our work provides important insight into the mechanisms underlying conditional cooperativity.

The vulnerable versatility of Salmonella antibiotic persisters during infection.

Tolerance and persistence are superficially similar phenomena by which bacteria survive bactericidal antibiotics. It is assumed that the same physiology underlies survival of individual tolerant and persistent bacteria. However, by comparing tolerance and persistence during Salmonella Typhimurium infection, we reveal that these two phenomena are underpinned by different bacterial physiologies. Multidrug-tolerant mutant Salmonella enter a near-dormant state protected from immune-mediated genotoxic damages. However, the numerous tolerant cells, optimized for survival, lack the capabilities necessary to initiate infection relapse following antibiotic withdrawal. In contrast, persisters retain an active state. This leaves them vulnerable to accumulation of macrophage-induced dsDNA breaks but concurrently confers the versatility to initiate infection relapse if protected by RecA-mediated DNA repair. Accordingly, recurrent, invasive, non-typhoidal Salmonella clinical isolates display hallmarks of persistence rather than tolerance during antibiotic treatment. Our study highlights the complex trade-off that antibiotic-recalcitrant Salmonella balance to act as a reservoir for infection relapse.

Alternative splicing of the Wnt trafficking protein, Wntless and its effects on protein-protein interactions.

BACKGROUND: Wntless (Wls) is a protein that regulates secretion of Wnt signaling molecules from Wnt-producing cells. Wnt signaling is known to be critical for several developmental and homeostatic processes. However, Wnt-independent functions of Wls are now being elucidated. Primates express an alternative splice variant of Wls (here termed WlsX). WlsX contains an alternatively spliced COOH-terminus, and does not appear to be able to sustain significant levels of WNT secretion because of its inability to undergo retrograde trafficking to the endoplasmic reticulum. The functional significance for this alternatively spliced form of Wls has not yet been elucidated. We previously identified a cohort of Wls interacting proteins using a combination of yeast 2-hybrid and candidate gene approaches. RESULTS: In the present study, we analyzed the interaction of WlsX with previously identified Wls interactors, and additionally screened for novel protein interactors of WlsX utilizing a membrane yeast two hybrid screen. Three novel Wls interactors, Glycoprotein M6A (GPM6A), Alkylglycerol Monooxygenase (AGMO), and ORAI1 were identified. Each of these novel WlsX interactors, as well as all other Wls interacting proteins identified previously, with the exception of the mu-opioid receptor, were found to interact with both Wls and WlsX splice forms. We show that WlsX can form homodimers, but that WlsX may not interact with Wls. CONCLUSIONS: WlsX has the ability to form homodimers and to interact with most known Wls interacting proteins. Taken together, our results suggest that Wls and WlsX may have overlapping, but distinct functions, including sensitivity to opioid drugs. While studies have focused on the ability of Wls interacting proteins to affect Wnt secretion, future efforts will explore the reciprocal regulation of these proteins by Wls, possibly via Wnt-independent mechanisms.