Mycobacterium avium-intracellulare complex organisms are the most common cause of mycobacterial lung disease other than tuberculosis and are the leading cause of morbidity and mortality in the HIV-infected AIDS patients. Infections caused by M. avium patients with AIDS often go untreated as existing antibiotics are ineffective in adequately controlling these infections. This necessitates the need for novel molecular targets for chemotherapy. Replication of the bacterium leading to its multiplication is one of the necessary steps to establish an infection. Initiation is the first committed step in the replication, and the replication process is believed to be regulated at the level of initiation. Thus, understanding the basic steps involved in initiation of DNA replication in M. avium will help define important molecular targets against which new generation drugs can be developed. Initiation of replication occurs once per cell cycle at a specific site on the chromosome called oriC. Initiation is believed to be triggered by the interactions of dnaA with its recognition sequences present in oriC called the dnaA-boxes. These interactions are thought to facilitate recruiting of other proteins resulting in the completion of initiation. This research proposal focuses on identification and characterization of the oriC and Dna protein of M. avium. To obtain oriC, chromosomal DNA fragments of M. avium that support autonomous replication when present in nonreplicative plasmids will be identified, cloned and their nucleotide sequence will be determined. Sequential deletions from both the 5' and 3' regions will be carried out to identify the minimal DNA region that is essential for oriC activity. Site directed mutagenesis will be carried out to identify the putative DnaA boxes that are essential for oriC activity. The ability of M. avium oriC to function as autonomously replicating sequences in other bacteria will be determined. The dnaA gene will be over-expressed, the gene product will be purified and its interaction with the wild type and mutant sequences will be investigated. Antisense dnaA oligonucleotides that target to the dnaA mRNA to affect the expression of M. avium dnaA gene will be explored in an effort to evaluate the role of the M. avium gene. Using oriC plasmids and cell free extracts, an in vitro replication system will be established. The ultimate long-term goal of these experiments is to utilize the knowledge thus gained to develop rational drugs that affect the initiation of replication thereby preventing growth and resulting M. avium infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041406-02
Application #
2673005
Study Section
AIDS and Related Research Study Section 5 (ARRE)
Project Start
1997-08-01
Project End
2001-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Health Center at Tyler
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
City
Tyler
State
TX
Country
United States
Zip Code
75708
Roberts, Gretta; Vadrevu, Indumathi S; Madiraju, Murty V et al. (2011) Control of CydB and GltA1 expression by the SenX3 RegX3 two component regulatory system of Mycobacterium tuberculosis. PLoS One 6:e21090
Madiraju, Murty; Madiraju, Sai Chandanda; Yamamoto, Kohji et al. (2011) Replacement of Mycobacterium smegmatis dnaA gene by Mycobacterium tuberculosis homolog results in temperature sensitivity. Tuberculosis (Edinb) 91 Suppl 1:S136-41
Maloney, Erin; Madiraju, Murty; Rajagopalan, Malini (2009) Overproduction and localization of Mycobacterium tuberculosis ParA and ParB proteins. Tuberculosis (Edinb) 89 Suppl 1:S65-9
Kiran, Manjot; Chauhan, Ashwini; Dziedzic, Renata et al. (2009) Mycobacterium tuberculosis ftsH expression in response to stress and viability. Tuberculosis (Edinb) 89 Suppl 1:S70-3
Maloney, Erin; Stankowska, Dorota; Zhang, Jian et al. (2009) The two-domain LysX protein of Mycobacterium tuberculosis is required for production of lysinylated phosphatidylglycerol and resistance to cationic antimicrobial peptides. PLoS Pathog 5:e1000534
Nair, Naveen; Dziedzic, Renata; Greendyke, Rebecca et al. (2009) Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants. Mol Microbiol 71:291-304
Kiran, Manjot; Maloney, Erin; Lofton, Hava et al. (2009) Mycobacterium tuberculosis ftsZ expression and minimal promoter activity. Tuberculosis (Edinb) 89 Suppl 1:S60-4
Yamamoto, Kohji; Moomey, Meredith; Rajagopalan, Malini et al. (2008) Facilitation of dissociation reaction of nucleotides bound to Mycobacterium tuberculosis DnaA. J Biochem 143:759-64
Chauhan, Ashwini; Lofton, Hava; Maloney, Erin et al. (2006) Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase. Mol Microbiol 62:132-47
Madiraju, Murty V V S; Moomey, Meredith; Neuenschwander, Pierre F et al. (2006) The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC. Mol Microbiol 59:1876-90

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