Functional analysis and structural investigations of MTB DosS sensory domain.

Mycobacterium tuberculosis (MTB) is a very successful pathogen, causing the deaths of approximately two million people a year world-wide. Survival of the pathogen in vivo is dependant on its ability to respond and adapt to changes within its environment. One method of adaptation is through two-component signal transduction systems, the phosphotransfer pathways that couple stimuli to responses. Generic two-component systems involve two conserved elements, a membrane bound histidine kinase, which is the sensory protein, and a response-regulator protein that controls the response usually by altering the expression of genes required for adaptive responses. The aim of this thesis is to investigate the structure and function of the sensory protein DosS (DevS). DosS is induced by exposure to hypoxia, NO and ethanol and is the only one of the 11 paired MTB two component systems for which inducers have been identified but the precise chemical nature of the signal is unknown. The N-terminal input region of the DosS sensor contains two putative GAF domains. Various fragments of the N-terminal region were cloned, expressed and purified to homogeneity. Ultraviolet-Visible spectral analysis reveals that full length DosS binds a classical high spin b haem cofactor. Mutagenesis identified histidine 149 of DosS, which is within the N-terminal GAF domain, as critical to haem-binding. This is the first known GAF domain to bind haem and the presence of a haem co-factor is consistent with the postulated involvement of DosS in oxygen and redox sensing. Based on this data a model for histidine kinase activation is suggested. The second GAF domain of DosS was analysed using NMR spectroscopy. Triple resonance NMR experiments enabled the identification and sequential assignment for 99 out of the 141 backbone amide proton and nitrogen resonances. In addition, both of the GAF domains were also subjected to protein crystallisation while the full length DosS was investigated using electron microscopy

A new family of periplasmic-binding proteins that sense arsenic oxyanions

Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that's role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate

Mycobacterium tuberculosis Transcriptional Adaptation, Growth Arrest and Dormancy Phenotype Development Is Triggered by Vitamin C

BACKGROUND: Tubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O(2) depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O(2) and rapidly induce the DevR regulon at an estimated O(2) saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Vitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a 'dormant' drug-tolerant phenotype, but in a manner independent of the DevRS/DosT system. The 'AA-dormancy infection model' offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-'dormant' M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB

Unified Nomenclature for Genes Involved in Prokaryotic Aerobic Arsenite Oxidation

International audienc