[Show abstract][Hide abstract] ABSTRACT: Protection against oxidative stress is one of the primary defense mechanisms contributing to the survival of Mycobacterium tuberculosis in the host. In this study, we provide evidence that OxyS, a LysR-type transcriptional regulator functions as an oxidative stress response regulator in mycobacteria. Overexpression of OxyS lowers expression of the catalase-peroxidase (KatG) gene in M. smegmatis. OxyS binds directly with the katG promoter region and a conserved, GC-rich T-N(11)-A motif for OxyS binding was successfully characterized in the core binding site. Interestingly, the DNA-binding activity of OxyS was inhibited by H(2)O(2), but not by dithiothreitol. Cys25, which is situated at the DNA-binding domain of OxyS, was found to have a regulatory role for the DNA-binding ability of OxyS in response to oxidative stress. In contrast, the other three cysteine residues in OxyS do not appear to have this function. Furthermore, the mycobacterial strain over-expressing OxyS had a higher sensitivity to H(2)O(2). Thus, OxyS responds to oxidative stress through a unique cysteine residue situated in its DNA-binding domain and negatively regulates expression of the katG gene. These findings uncover a specific regulatory mechanism for mycobacterial adaptation to oxidative stress.
PLoS ONE 01/2012; 7(1):e30186. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Confident protein-DNA interaction (PDI) data could significantly improve our understanding of transcriptional regulation network in both prokaryotes and eukaryotes. New methods need to be established for validating and mining the protein-DNA interaction data produced by bioinformatic tools and large-scale screening assays. In this study, we integrated bacterial one-hybrid technique (B1H) with classical chromatin immunoprecipitation (ChIP) assay to develop an innovative B1H-ChIP method, which has the advantages of being high-throughput, low cost, and easy-to-perform. Using this method, we validated two pairs of previously reported PDIs and further successfully discovered five novel target genes for Mce2R and four novel regulators of the gene dnaA in the human pathogen Mycobacterium tuberculosis. New PDI data suggest that Mce2R may play novel roles in the regulation of multi-drug resistance, cell wall synthesis, and intracellular growth of M. tuberculosis, and there exists a probable selective regulation of dnaA under different host conditions. Our findings provide important new information for understanding unique regulatory mechanisms in the pathogen. The B1H-ChIP approach has wide applications both in validating and discovering PDIs and in unraveling transcriptional regulatory network in prokaryotes and eukaryotes.
Applied Microbiology and Biotechnology 12/2011; 93(3):1257-69. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Virulence in pathogenic bacteria is due in part to the action of two-component systems. However, in the human pathogen Mycobacterium tuberculosis, the molecular mechanisms underlying these systems are as yet unclear. In this study, MtrA was shown to contain a functional C-terminus and also to have Ca(2+) as its preferred cofactor for DNA binding. Further mutation experiments demonstrated that the C-terminus of MtrA was responsible for specific interactions with the target DNA motif and also with its partner protein, MtrB. The physical interaction between MtrA and MtrB inhibited DNA binding by MtrA. These findings yield critical information about the unique regulatory mechanisms of the essential MtrAB two-component system in this pathogen.
Journal of Biochemistry 11/2010; 148(5):549-56. · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Analysis of the protein-protein interaction network of a pathogen is a powerful approach for dissecting gene function, potential signal transduction, and virulence pathways. This study looks at the construction of a global protein-protein interaction (PPI) network for the human pathogen Mycobacterium tuberculosis H37Rv, based on a high-throughput bacterial two-hybrid method. Almost the entire ORFeome was cloned, and more than 8000 novel interactions were identified. The overall quality of the PPI network was validated through two independent methods, and a high success rate of more than 60% was obtained. The parameters of PPI networks were calculated. The average shortest path length was 4.31. The topological coefficient of the M. tuberculosis B2H network perfectly followed a power law distribution (correlation = 0.999; R-squared = 0.999) and represented the best fit in all currently available PPI networks. A cross-species PPI network comparison revealed 94 conserved subnetworks between M. tuberculosis and several prokaryotic organism PPI networks. The global network was linked to the protein secretion pathway. Two WhiB-like regulators were found to be highly connected proteins in the global network. This is the first systematic noncomputational PPI data for the human pathogen, and it provides a useful resource for studies of infection mechanisms, new signaling pathways, and novel antituberculosis drug development.
Journal of Proteome Research 10/2010; 9(12):6665-77. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) remains to be a major infectious disease throughout the world. However, the current vaccine for TB has variable protective efficacy, and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use. One of the potential strategies in developing a new diagnostic method and in improving the TB vaccine involves the identification of novel antigenic candidates. This paper aims to identify systematically the novel antigenic proteins with the greatest potential as protective or diagnostic antigens by using the differential response of Mycobacterium tuberculosis proteins to serum from TB patients and healthy individuals. Approximately 87% of the open reading frames of M. tuberculosis were successfully cloned into IPTG-inducible expression vectors. The clone sets were expressed in Escherichia coli, purified under denatured conditions, and tested for antigenicity using a mixture of sera from 15 TB patients. Out of the 3480 proteins screened, 249 proteins had significant reactions with the serum samples. Among the 249 proteins, 20 proteins were identified as most reactive. Compared with the commercial test kits, 3 novel antigens from the top 20 proteins, namely, Rv1987, Rv3807c, and Rv3887c, provided better sensitivity and accuracy. These newly identified antigenic proteins may be used as candidates for serodiagnostic application and vaccine development. Overall, this study's findings may serve as an essential reference for developing new TB diagnostic methods and more effective tuberculosis vaccines.
Journal of Proteome Research 09/2010; 9(9):4812-22. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The two-component systems of Mycobacterium tuberculosis are apparently required for its growth and resistance in hostile host environments. In such environments, MtrAB has been reported to regulate the expression of the M. tuberculosis replication initiator gene, dnaA. However, the dnaA promoter binding sites and many potential target genes for MtrA have yet to be precisely characterized.
In this study, a 7 bp sequence motif in the dnaA promoter region was identified for MtrA binding using DNaseI footprinting assays and surface plasmon resonance (SPR) analysis. Approximately 420 target genes potentially regulated by MtrA, including the isoniazid inducible gene iniB, were further characterized from M. tuberculosis and M. smegmatis genomes. When assayed using quantitative real-time PCR (qRT-PCR), many of the target genes demonstrated significant expression changes when the antisense mRNA of the mtrA gene was expressed in M. smegmatis. The recombinant mycobacteria grew in length and were more sensitive to two anti-tuberculosis drugs, isoniazid and streptomycin.
These findings yield critical information about the regulatory mechanisms of the MtrAB two-component system and its role in the drug resistance of M. smegmatis.
[Show abstract][Hide abstract] ABSTRACT: Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms.
Protein Expression and Purification 09/2009; 69(1):47-53. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sequence-specific DNA-binding transcription factors have widespread biological significance in the regulation of gene expression. However, in lower prokaryotes and eukaryotic metazoans, it is usually difficult to find transcription regulatory factors that recognize specific target promoters. To address this, we have developed in this study a new bacterial one-hybrid reporter vector system that provides a convenient and rapid strategy to determine the specific interaction between target DNA sequences and their transcription factors. Using this system, we have successfully determined the DNA-binding specificity of the transcription regulator Rv3133c to a previously reported promoter region of the gene Rv2031 in Mycobacterium tuberculosis. In addition, we have tested more than 20 promoter regions of M. tuberculosis genes using this approach to determine if they interact with approximately 150 putative regulatory proteins. A variety of transcription factors are found to participate in the regulation of stress response and fatty acid metabolism, both of which comprise the core of in vivo-induced genes when M. tuberculosis invades macrophages. Interestingly, among the many new discovered potential transcription factors, the WhiB-like transcriptional factor WhiB3 was identified for the first time to bind with the promoter sequences of most in vivo-induced genes. Therefore, this study offers important data in the dissection of the transcription regulations in M. tuberculosis, and the strategy should be applicable in the study of DNA-binding factors in a wide range of biological organisms.
Genome Research 03/2009; 19(7):1301-8. · 14.40 Impact Factor