Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.     

Streptomyces coelicolor Dps-like proteins: differential dual roles in response to stress during vegetative growth and in nucleoid condensation during reproductive cell division

The Dps protein, a member of the ferritin family, contributes to DNA protection during oxidative stress and plays a central role in nucleoid condensation during stationary phase in unicellular eubacteria. Genome searches revealed the presence of three Dps-like orthologues within the genome of the Gram-positive bacterium Streptomyces coelicolor . Disruption of the S. coelicolor dpsA , dpsB and dpsC genes resulted in irregular condensation of spore nucleoids in a gene-specific manner. These irregularities are correlated with changes to the spacing between sporulation septa. This is the first example of these proteins playing a role in bacterial cell division. Translational fusions provided evidence for both developmental control of DpsA and DpsC expression and their localization to sporogenic compartments of aerial hyphae. In addition, various stress conditions induced expression of the Dps proteins in a stimulus-dependent manner in vegetative hyphae, suggesting stress-induced, protein-specific protective functions in addition to their role during reproductive cell division. Unlike in other bacteria, the S. coelicolor Dps proteins are not induced in response to oxidative stress.     

Matrix-assisted refolding and redox properties of WhiB3/Rv3416 of Mycobacterium tuberculosis H37Rv

Redox stress is one of the major challenges faced by Mycobacterium tuberculosis during early infection and latency. The mechanism of sensing and adaptation to altered redox conditions is poorly understood. whiB family of Mtb is emerging as an important class of stress responsive genes. WhiB3/Rv3416 has been shown to be important for pathogenesis in animal model and was recently shown to co-ordinate a Fe-S cluster. Here, we report a simple, rapid and efficient matrix-assisted refolding method and important redox properties of WhiB3. Similar to other WhiB proteins, WhiB3 also has four conserved cysteine residues, where two of them are present in a CXXC motif. The Fe-S cluster of WhiB3 remained bound in the presence of strong protein denaturant. Upon cluster removal due to oxidation, the four cysteine residues which are ligands of Fe-S cluster, formed two intra-molecular disulfide bridges where one of them is possibly between the cysteines of CXXC motif, an important feature of several thiol-disulfide oxido-reductases. Far-UV CD spectroscopy revealed the presence of both alpha-helices and beta-strands in apo WhiB3. The secondary structural elements of apo WhiB3 were found resistant for thermal denaturation. The results demonstrated that apo WhiB3 functions as a protein disulfide reductase similar to thioredoxins. The importance of WhiB3 in redox sensing and its possible role in mycobacterial physiology has been discussed.     

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.     

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E_MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E_MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E_MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.     

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.