Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS 201 ) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His 6 -tagged proteins from Escherichia coli.

No abstract

BackgroundAggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine.MethodologyWe have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Aβ1-42) peptide is modified. Changes in the hydrodynamic volume of Aβ1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Aβ1-42. Arginine increases the solubility of Aβ1-42 peptide in aqueous medium. It decreases the aggregation of Aβ1-42 as observed by atomic force microscopy.ConclusionsBased on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

No abstract

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR: :kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-tocell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen ; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.

The modified Poisson-Boltzmann theory is applied to a study of the cell model of linear polyelectrolyte solutions. The theoretical results for polyion-simple ion correlations are compared with those obtained in parallel Monte Carlo simulations and classical Poisson-Boltzmann calculations for the same model. The modified Poisson-Boltzmann predictions are found to be in complete agreement with the simulation data for monovalent simple ions and moderate concentrations, while the Poisson-Boltzmann theory is valid at low concentrations. In solutions containing divalent ions, where standard theories tend to give a very rough estimate of the simulated structure, the modified Poisson-Boltzmann results remain very close to those obtained from the simulations. The theory appears to provide an essentially correct account of interionic correlations, which are neglected in mean field theory, such as the Poisson-Boltzmann approximation, leading to poor results. The calculations for high ionic strengths illustrate the limitations of the cell model due to the neglect of the correlations between double layers surrounding neighboring polyions in solution.

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3 H-labeled and 14 C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

Mycobacterium tuberculosis faces various stressful conditions inside the host and responds to them through a coordinated regulation of gene expression. We had previously reported identification of the virS gene of M. tuberculosis (Rv3082c) belonging to the AraC family of transcriptional regulators. In the current study, we show that the seven genes (Rv3083-Rv3089) which are present divergently to virS (Rv3082c) constitute an operon designated the mymA operon. Further investigation on the regulation of this operon showed that transcription of the mymA operon is dependent on the presence of VirS protein. A four-fold induction of the mymA operon promoter occurs specifically in wild-type M. tuberculosis and not in the virS mutant of M. tuberculosis (MtbDeltavirS) when exposed to acidic pH. Expression of the mymA operon was also induced in infected macrophages by 10-fold over a 6-day period. To gain an insight into the function of the proteins encoded by this operon, we carried out a bioinformatic analysis, which suggested the involvement of these proteins in the modification of fatty acids required for cell envelope. This was supported by altered colony morphology and cell envelope structure displayed by the virS mutant of M. tuberculosis (MtbDeltavirS).