RESUMEN
Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Glutatión/metabolismo , Espacio Intracelular/metabolismo , Espacio Intracelular/microbiología , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Regulación Alostérica/efectos de los fármacos , Proteínas Bacterianas/metabolismo , ADN/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glutatión/farmacología , Espacio Intracelular/efectos de los fármacos , Listeria monocytogenes/efectos de los fármacos , Macrófagos/metabolismo , Mutación/genética , Factores de Terminación de Péptidos/metabolismo , Unión Proteica , Selección Genética/genética , Supresión Genética/genética , Virulencia/genéticaRESUMEN
Hydrogen peroxide (H(2)O(2)) is a critically important signaling molecule. Endogenous H(2)O(2) mediates diverse physiological processes both intra- and intercellularly; and enzymatically generated H(2)O(2) is a widely used reporter molecule at biosensors that rely on enzymes to detect non-electroactive species. However, the development and application of electroanalytical methods for the direct detection of this molecule has been challenging because the electron transfer kinetics for the irreversible oxidation of H(2)O(2) are slow. We comparatively characterize the electrochemical oxidation of H(2)O(2) on bare and Nafion(®)-coated platinum and carbon-fiber microdisc electrodes using fast-scan cyclic voltammetry (FSCV). Using a waveform ranging from +0.2 to +1.3 V at 400 V s(-1), the electrocatalytic properties of the platinum surface were not readily apparent, and the carbon-fiber microelectrode demonstrated greater sensitivity and selectivity toward H(2)O(2). Nafion(®)-coating further enhanced detection on carbon electrodes. These results confirm that platinum electrodes, with or without Nafion(®), will not work acceptably with this approach, and confirm the value of carbon-fiber microelectrodes relative to more traditionally used platinum electrodes in the direct detection of rapid H(2)O(2) fluctuations using FSCV.