Research Projects: Mycobacterium tuberculosis
Overview | Project page
ASU | School of Life Sciences | Biodesign Institute
Overview | Project page
click on diagrams for larger image
To maintain its status as an extraordinary successful human pathogen, Mycobacterium tuberculosis must adapt to and survive a multitude of external stresses experienced within the body and during transmission. These cellular responses are often mediated by two-component regulators which have unequivocally been implicated in the disease processes of this human pathogen. Our long-term goal is to determine the role of two-component regulators in mediating the dynamic range of environments and stresses encountered during M. tuberculosis host aerosolization, intracellular survival, active disease, persistence, and transition from a persistent infection to an active disease state and to identify the regulated gene products that are required for virulence.
Identification of trcRS-regulated genes is ongoing and is being performed using transcriptomic and proteomic analyses and various genetic and biochemical approaches. The results of these experiments will be subsequently validated using quantitative RT-PCR and additional studies to demonstrate indirect or direct regulation. Identified trcRS-regulated genes will then be mutagenized and investigated in virulence studies in macrophages and mice.
We have found that TrcR binds its own promoter via an AT-rich sequence and that phosphorylated TrcR exhibits an increased binding affinity. In addition, trcR expression is autoregulated in E. coli, and the AT-rich sequence is required for autoregulation. These experiments have proven to be useful in identifying another gene, rv1057, that is bound by TrcR via AT-rich sequences and repressed by TrcR. The rv1057 gene is also expressed during M. tuberculosis growth in macrophages, and the protein appears to be a seven-bladed β-propeller of unknown function.
As an identified trcR-repressed gene, experiments to determine the function and structure of rv1057 are being performed. rv1057 is expressed in human and murine macrophages and under in vitro growth conditions that mimic an intraphagosomal environment. We have already constructed an M. tuberculosis rv1057 mutant which will be subjected to virulence studies in macrophages and mice. As a gene expressed during M. tuberculosis intracellular growth and a protein with a unique predicted structure, X-ray crystallography studies to determine its high-resolution structure are currently underway in collaboration with Dr. Rebekka Wachter, Dept. of Chemistry and Biochemistry at ASU.
click on diagram for larger image
The rv1626 response regulator gene is expressed after 18, 48, and 110 h of M. tuberculosis intracellular growth, while expression of the rv3143 orphan response regulator gene was not detected during M. tuberculosis growth in human macrophages. However, in another study, the rv3143 gene exhibits a similar expression profile in macrophages to the mprA response regulator gene which is required for maintaining a persistent M. tuberculosis infection. Recently constructed rv1626 and rv3143 mutants will both be analyzed using the methods described above for the trcRS mutant to determine the regulons and their roles in M. tuberculosis pathogenesis and/or physiology.
The high resolution crystal structure of Rv1626 demonstrates that the N-terminus harbors a prototypical receiver domain with a C-terminal domain exhibiting structural homology to a unique RNA binding domain involved in transcriptional antitermination (Morth et al. 2004). Demonstrated phosphorylation relay between the Rv3220c orphan histidine kinase and Rv1626 establishes these proteins as a two-component regulatory circuit (Morth et al. 2005). Due to the unique putative function, Rv1626 is designated a phosphorylation-dependent transcriptional antitermination regulator (PdtaR) and its cognate histidine kinase, Rv3220c, a phosphorylation-dependent transcriptional antitermination sensor (PdtaS). Efforts are underway to understand how this unique response regulator controls transcription via antitermination and its role in M. tuberculosis pathogenesis.
Crystal structure of the PdtaR transcriptional antiterminator (A)
and the superimposed ANTAR domains of PdtaR (red) and
Pseudomonas aeruginosa AmiR (cyan) (Morth et al. 2004).
The prrAB two-component system is expressed by M. tuberculosis after 48 h of growth in human macrophages, while the mtrAB is constitutively expressed during M. tuberculosis growth in human macrophages. The inability to generate prrAB and mtrAB mutants strongly suggests that these systems are essential to M. tuberculosis viability. A multi-faceted approach involving the generation of single amino acid mutations, antisense constructs, and a reporter library is underway for understanding the molecular basis of prrAB and mtrAB transcriptional regulation and for deciphering their roles in mycobacterial virulence. Additionally, we plan to use use proteomic and modeling approaches to identify inhibitors of these regulators.
