Bacille Calmette-Guérin (BCG) vaccines are live attenuated strains of Mycobacterium bovis administered to prevent tuberculosis. To better understand the differences between M. tuberculosis, M. bovis, and the various BCG daughter strains, their genomic compositions were studied by performing comparative hybridization experiments on a DNA microarray. Regions deleted from BCG vaccines relative to the virulent M. tuberculosis H37Rv reference strain were confirmed by sequencing across the missing segment of the H37Rv genome. Eleven regions (encompassing 91 open reading frames) of H37Rv were found that were absent from one or more virulent strains of M. bovis. Five additional regions representing 38 open reading frames were present in M. bovis but absent from some or all BCG strains; this is evidence for the ongoing evolution of BCG strains since their original derivation. A precise understanding of the genetic differences between closely related Mycobacteria suggests rational approaches to the design of improved diagnostics and vaccines.
Few tools exist to assess replication of chronic pathogens during infection. This has been a considerable barrier to understanding latent tuberculosis, and efforts to develop new therapies generally assume that the bacteria are very slowly replicating or nonreplicating during latency [1][2][3] . To monitor Mycobacterium tuberculosis replication within hosts, we exploit an unstable plasmid that is lost at a steady, quantifiable rate from dividing cells in the absence of antibiotic selection. By applying a mathematical model, we calculate bacterial growth and death rates during infection of mice. We show that during chronic infection the cumulative bacterial burden-enumerating total live, dead and removed organisms encountered by the mouse lung-is substantially higher than estimates from colony forming units. Our data show that M. tuberculosis replicates throughout the course of chronic infection of mice and is restrained by the host immune system. This approach may also shed light on the replication dynamics of other chronic pathogens.Many deadly diseases, such as AIDS, malaria and tuberculosis, result from infections that persist for long periods. Disease processes that unfold over months or years are especially difficult to model in the laboratory. New approaches are sorely needed to generate insight into the pathogenesis and treatment of such agents. 8,[11][12][13] . Additionally, isoniazid, the first-line agent used to treat latent tuberculosis, has virtually no efficacy against nonreplicating bacilli in vitro [14][15][16] or in vivo 17 . Similarly, the rapid reactivation of latent Mtb after anti-tumor necrosis factor-α therapy 18 seems inconsistent with infection by bacteria in a nonreplicating state.To reassess mycobacterial replication, we developed a method based on pBP10, a circular plasmid carrying a kanamycin resistance marker 19 . Plasmids replicate coordinately with bacterial DNA, sometimes requiring antibiotic selection for their maintenance. Without selection, plasmids are lost from a proportion of daughter cells during cell division, and thus decreasing plasmid numbers can act as a marker of division. We introduced pBP10 into Mycobacterium smegmatis and Mtb to evaluate the feasibility of using this plasmid as a replication clock.M. smegmatis-pBP10 was maintained in log-phase culture under conditions where doubling times ranged from 2 h to 5 h. Plasmid loss was determined by plating on agar with and without kanamycin. The plasmid was steadily lost as cells divided, with a rate proportional to the growth rate under each condition (Fig. 1a,b). The plasmid loss per generation was constant, regardless of the replication rate (Fig. 1c). We applied a mathematical model (Supplementary Methods online) to determine the segregation constant s -the frequency of daughter cells losing plasmid per generation. This value (0.11 ± 0.0074 (s.d.)) was reproducible across all tested growth conditions.Mtb-pBP10 was maintained in log phase for ~30 generations by subculturing every 2-3 d without antibiotics unde...
Recently, cholesterol was identified as a physiologically important nutrient for Mycobacterium tuberculosis survival in chronically infected mice. However, it remained unclear precisely when cholesterol is available to the bacterium and what additional bacterial functions are required for its metabolism. Here, we show that the igr locus, which we previously found to be essential for intracellular growth and virulence of M. tuberculosis, is required for cholesterol metabolism. While igr-deficient strains grow identically to the wild type in the presence of short-and long-chain fatty acids, the growth of these bacteria is completely inhibited in the presence of cholesterol. Interestingly, this mutant is still able to respire under cholesterol-dependent growth inhibition, suggesting that the bacteria can metabolize other carbon sources during cholesterol toxicity. Consistent with this hypothesis, we found that the growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as mutation of the mce4 sterol uptake system partially suppresses this effect. In addition, the ⌬igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout infection.
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