Abstract
An experimental system of Mycobacterium tuberculosis growth) in a carbon-limited chemostat, has been established using Mycobacterium bovis BCG as a model organism. In this model carbon- limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 hours (D = 0.03 h-1) was adopted to represent the acute phase of infection whereas a slower dilution rate, equivalent to a doubling time of 69 hours (D = 0.01 h-1) was used to model mycobacterial persistence. Cell macromolecular (RNA, DNA, carbohydrate and lipid) and elemental (C, H, N) compositions of biomass were determined in the model, revealing an atypical bacterial cell containing large amounts of lipid and carbohydrate. The correlation of RNA content with growth rate indicated that ribosome production in carbon limited M. bovis BCG cells is subject to growth rate dependent control. These results also clearly show that the proportion of lipid in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. This may have particular significance for mycobacteria during chronic TB as this stage of the disease is thought to involve a metabolic switch in the bacteria's carbon source to fats. Given the importance of isocitrate lyase in fat metabolism and the survival of persistent bacteria, an isocitrate lyase mutant of M. bovis BCG was constructed and used as a biological probe to test the validity of the chemostat model of TB persistence. The mutant strain behaved identically to the wild type at the fast growth rate but was attenuated for survival at the slower growth rate demonstrating that isocitrate lyase is important for survival in the model. Transcriptomic analysis using DNA microarrays demonstrated that 353 genes were differentially expressed between the slow and fast growing mycobacterial cells. These data show a significant shift in metabolism towards scavenging nutrients from the environment and the upregulation of several "persistence genes" in the slow growth rate. Optimization of two-dimensional electrophoresis methodology allowed the cellular protein composition of BCG in the model to be studied. Expression of 7 proteins increased and 3 proteins decreased in the slow relative to the faster growing chemostat culture. Proteins identified included the ATP-binding protein Rv2623 and Rv2626c, proteins known to be upregulated during growth in vivo. These studies demonstrated the power of combining chemostat models with global system approaches such as proteomics, transcriptomics and fluxomics technologies in deciphering the complex programme of M. tuberculosis during persistence.