Toxin-mediated depletion of NAD and NADP drives persister formation in a human pathogen.

Toxin-antitoxin (TA) systems are widespread in bacteria and implicated in genome stability, virulence, phage defense, and persistence. TA systems have diverse activities and cellular targets, but their physiological roles and regulatory mechanisms are often unclear. Here, we show that the NatR-NatT TA system, which is part of the core genome of the human pathogen Pseudomonas aeruginosa, generates drug-tolerant persisters by specifically depleting nicotinamide dinucleotides. While actively growing P. aeruginosa cells compensate for NatT-mediated NAD+ deficiency by inducing the NAD+ salvage pathway, NAD depletion generates drug-tolerant persisters under nutrient-limited conditions. Our structural and biochemical analyses propose a model for NatT toxin activation and autoregulation and indicate that NatT activity is subject to powerful metabolic feedback control by the NAD+ precursor nicotinamide. Based on the identification of natT gain-of-function alleles in patient isolates and on the observation that NatT increases P. aeruginosa virulence, we postulate that NatT modulates pathogen fitness during infections. These findings pave the way for detailed investigations into how a toxin-antitoxin system can promote pathogen persistence by disrupting essential metabolic pathways.

A genetic switch controls Pseudomonas aeruginosa surface colonization.

Efficient colonization of mucosal surfaces is essential for opportunistic pathogens like Pseudomonas aeruginosa, but how bacteria collectively and individually adapt to optimize adherence, virulence and dispersal is largely unclear. Here we identified a stochastic genetic switch, hecR-hecE, which is expressed bimodally and generates functionally distinct bacterial subpopulations to balance P. aeruginosa growth and dispersal on surfaces. HecE inhibits the phosphodiesterase BifA and stimulates the diguanylate cyclase WspR to increase c-di-GMP second messenger levels and promote surface colonization in a subpopulation of cells; low-level HecE-expressing cells disperse. The fraction of HecE+ cells is tuned by different stress factors and determines the balance between biofilm formation and long-range cell dispersal of surface-grown communities. We also demonstrate that the HecE pathway represents a druggable target to effectively counter P. aeruginosa surface colonization. Exposing such binary states opens up new ways to control mucosal infections by a major human pathogen.

Filling of a water-free void explains the allosteric regulation of the ß1-adrenergic receptor by cholesterol.

Recent high-pressure NMR results indicate that the preactive conformation of the ß1-adrenergic receptor (ß1AR) harbours completely empty cavities of ~100 Å3 volume, which disappear in the active conformation of the receptor. Here we have localized these cavities using X-ray crystallography of xenon-derivatized ß1AR crystals. One of the cavities is in direct contact with the cholesterol-binding pocket. Solution NMR shows that addition of the cholesterol analogue cholesteryl hemisuccinate impedes the formation of the active conformation of detergent-solubilized ß1AR by blocking conserved G protein-coupled receptor microswitches, concomitant with an affinity reduction of both isoprenaline and G protein-mimicking nanobody Nb80 for ß1AR detected by isothermal titration calorimetry. This wedge-like action explains the function of cholesterol as a negative allosteric modulator of ß1AR. A detailed understanding of G protein-coupled receptor regulation by cholesterol by filling of a dry void and the easy scouting for such voids by xenon may provide new routes for the development of allosteric drugs.