The primary goal of our research is the integration of functional genomics with molecular genetics and biochemistry to understand bacterial virulence mechanisms. Our work currently focuses on Mycobacterium tuberculosis, the causative agent of tuberculosis.

Among communicable diseases, TB is the second leading cause of death worldwide (Frieden et al. 2003). The most frequent form of TB is latent and does not cause clinical symptoms. Latent TB leads to active TB in ~10% of the immune-competent and 50-80% of immune-compromised individuals. Drug-sensitive TB can be effectively cured by treating patients for 6-9 months with multiple drugs (Mitchison, 1980; McKinney, 2000). Multiple drugs are most likely necessary to kill bacterial populations of different drug tolerance that seem to co-exist in vivo. The prolonged treatment seems necessary to kill slowly replicating or non-replicating bacteria that are only infrequently susceptible to treatment. Shortening the duration of chemotherapy - for example due to limited drug supply or patient non-compliance - selects for drug-resistant and multiple drug resistant (MDR) TB. MDR-TB is difficult to treat, has a high mortality and can be rapidly fatal. The duration of antibiotic treatment necessary to prevent MDR-TB seems, therefore, to be the most serious shortfall of anti-TB chemotherapy. By integrating functional genomics with molecular genetics and biochemistry we hope to identify new drug targets that may allow the development of shorter anti-TB chemotherapies.

Our work initially focused on the characterization of the transcriptome of intraphagosomal M. tuberculosis (Ehrt et al. 2002; Schnappinger et al. 2003; Voskuil et al. 2003). Macrophages are central to the effector arm of immune defense against most long-lived, nonviral intracellular pathogens. Macrophages internalize microbes into phagosomes that undergo maturational events that expose the microbes to acid, lytic enzymes, oxygenated lipids, fatty acids and reactive oxygen and nitrogen intermediates. Despite this battery of antimicrobial molecules, some pathogens are able to survive and replicate within macrophages without escaping the phagosome. M. tuberculosis is among the microorganisms most successful at adapting to long-term residence in macrophage phagosomes. The phagosome is a difficult organelle to study because profound biochemical shifts accompany the host cell's effort to kill and degrade microbial pathogens. To identify biochemical conditions encountered within phagosomes we compared the transcriptomes of M. tuberculosis within macrophages with the transcriptomes of M. tuberculosis in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence suggest the phagosomal environment to be nitrosative, oxidative, functionally hypoxic, carbohydrate-poor and capable of perturbing the pathogen's cell envelope.

In collaboration with Dr. Ehrt (Department of Microbiology, Weill Medical College of Cornell University) we recently developed TetR-controlled expression systems that allow silencing of mycobacterial genes in vitro and during macrophage infections (manuscript in preparation). We are using these systems to determine the importance of genes that - based on our transcriptome studies - seem important for the survival of M. tuberculosis within the host. These studies should provide proof-of-principle for the hypotheses that TetR-controlled gene silencing can be used to identify biological processes that are essential for growth and persistence of mycobacteria during mouse infections, genetically mimic the action of antibiotics on the progression of M. tuberculosis infections and thus aid the prioritization of dug targets, and study the function of essential genes using functional genomics.

Ehrt, S. Voskuil, M.I., Schoonik, G.K. and Schnappinger, D. (2002) Genome-wide expression profiling of intracellular bacteria: The interaction of Mycobacterium tuberculosis with -macrophages. Methods in Microbiology 32, 169-179
Frieden, T. R., Sterling, T. R., Munsiff, S. S., Watt, C. J. & Dye, C. Tuberculosis. Lancet 362, 887-99 (2003).
McKinney, J. D. In vivo veritas: the search for TB drug targets goes live. Nat Med 6, 1330-3 (2000).
Mitchison, D. A. Treatment of tuberculosis. The Mitchell lecture 1979. J R Coll Physicians Lond 14, 91-5, 98-9 (1980).
Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D., Nathan, C., and Schoolnik, G. K. (2003). Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 198, 693-704.
Voskuil, M. I., Schnappinger, D., Visconti, K. C., Harrell, M. I., Dolganov, G. M., Sherman, D. R., and Schoolnik, G. K. (2003). Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198, 705-713.