igr Genes and Mycobacterium tuberculosis cholesterol metabolism

Abstract

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 Delta 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.

FIG. 1.

H37Rv:Δigr is unable to grow in the presence of cholesterol. Growth of H37Rv (▪), H37Rv:Δigr (•), and the complemented H37Rv:Δigr:igr strain (⧫) in MM (filled symbols in panel A) or in MM+G or MM+CG (open symbols in panels A and B, respectively). The data shown are the mean of triplicate cultures ± the standard deviation. For all panels, the data shown are from one of at least two experiments.

FIG. 2.

H37Rv:Δigr is not attenuated for ATP production or alamarBlue reduction in the presence of cholesterol. (A) Amount of ATP per cell as a percentage of H37Rv after 3 days in 7H9, MM, or MM+CG. (B) alamarBlue reduction as a percentage of H37Rv after 48 h. Representative data from one of three experiments are shown as the mean of triplicate cultures ± the standard deviation.

FIG. 3.

H37Rv:Δigr can import and partially degrade cholesterol. (A) The cholesterol molecule depicting C-4 and C-26. (B) CO₂ production measured by BACTEC for H37Rv and the Δigr, Δigr:igr, and Δmce4 mutant strains when incubated with ¹⁴C-4-radiolabeled cholesterol. (C, D) Total lipid fractions measuring incorporation of ¹⁴C-26-radiolabeled cholesterol by TLC (C) and counts per minute (D). (C) Lane 1, ¹⁴C-26-labeled cholesterol; lane 2, H37Rv; lane 3, Δigr mutant; lane 4, Δigr:igr mutant. Major band is sulfolipid 1 (identified using a purified standard and mass spectrometry by S. Gillmore).

FIG. 4.

Deletion of mce4 partially rescues cholesterol growth inhibition of Δigr. (A) Growth of H37Rv (▪), H37Rv:Δigr (•), and H37Rv:ΔigrΔmce4 (▴) strains in MM+CG. (B) Amount of ATP per cell as a percentage of H37Rv. (C) Killing of THP-1 cells by H37Rv (▪), H37Rv:Δigr (•), and H37Rv:ΔigrΔmce4 (▴) strains monitored by alamarBlue staining. Data shown are the mean and standard deviation of triplicate cultures. For all panels, data are representative of at least duplicate experiments.

FIG. 5.

Replication dynamics of H37Rv:Δigr in mice. (A). The fraction of the bacterial population carrying unstable plasmid pBP10 confirms that mutant strain H37Rv:Δigr replicates more slowly than the wild type in C57BL/6 mice. H37Rv (▪), H37Rv:Δigr (•). The number of CFU per lung (filled line, closed symbols) and the percentage of bacteria carrying plasmid (dashed line, open symbols) are shown. (B) Mice infected with strain H37Rv:Δigr have a lower cumulative bacterial burden than those infected with the wild type. The number of CFU per lung (filled line, filled symbols), the cumulative bacterial burden (CBB) (filled line, open symbols), and the 95% confidence interval (dashed line) are shown. The data shown are from a single experiment, the average from five infected mice per time point with the standard deviation, and the data from H37Rv-infected mice previously appeared in reference .

FIG. 6.

Mce4 deletion suppresses the in vivo growth defect of the Δigr mutant. CFU were counted at 21 days postinfection in both the lungs and spleens of mice infected with wild-type H37Rv or the Δigr or Δigr Δmce4 mutant strain. The results for the Δigr mutant are statistically significantly different from those for the Δigr Δmce4 mutant in both the lung (P = 0.0002) and the spleen (P = 0.05).