Mycobacterium tuberculosis is an exquisitely adapted human pathogen that infects roughly a third of the world's population. Like many other intracellular parasites, M. tuberculosis resides largely within a modified phagosomal compartment of the host macrophage during infection. While there are obvious advantages to this intracellular life-style, survival and replication within a host-derived membrane also pose several particular challenges. Perhaps the most fundamental of these is the need to acquire nutrients, for example, carbon, from the host cell. While all vacuolar pathogens face this challenge, it remains unclear which carbon sources are readily available and how they are extracted from the cell.
Host lipids have long been implicated as nutrient sources for M
during intracellular growth and chronic infection (4
). This was first suggested by the observation that fatty acids but not carbohydrates stimulate respiration of M
isolated from the mouse lung (1
). Subsequently, sequencing of the M
genome revealed at least 250 genes potentially involved in lipid metabolism (4
). Many of these genes are transcriptionally induced during intracellular growth, and a few are known to be required for infection (2
). However, the complexity of M
lipid metabolism has made it difficult to determine if any individual gene is genuinely required for host lipid catabolism, as opposed to the synthesis or modification of an endogenous bacterial lipid.
We previously identified a region of the M
genome (Rv3545c to Rv3540c) that is important for growth in macrophages and in mice (3
) and therefore was renamed igr
owth. A number of other studies have also indicated that these genes are important in vivo (16
). The convergence of these studies on this single locus highlights its potential importance, yet little was known about the mechanism by which these genes contribute to mycobacterial survival in the host. The igr
locus consists of a single operon containing genes for a putative cytochrome P450 (igrA
), two acyl coenzyme A dehydrogenases (igrBC
), a conserved hypothetical protein with a hot dog domain (igrD
), a putative enoyl coenzyme A hydratase with a hot dog domain (igrE
), and a lipid carrier protein (igrF
). Based on these homologies, we predicted that the igr
locus was involved in lipid metabolism, but its precise function remained unclear.
Recently, cholesterol has been implicated as significant for M
during chronic infection. This was first hypothesized based on synteny between a cholesterol-catabolic locus of Rhodococcus
strain RHA1, an environmental relative of M
, and a region of the mycobacterial chromosome known to be critical for bacterial growth in vivo (19
). Included in this region is the mce4
operon, which encodes an ABC-like transporter that represents the major cholesterol uptake system of the bacterium (12
). Mutants lacking the ability to import cholesterol have a specific defect in survival during the chronic phase of infection in mice and in the gamma interferon-activated macrophages that characterize this stage of disease (15
). Thus, cholesterol appears to be an important nutrient during chronic M
infection; however, it remains unclear if cholesterol is a constituent of the mycobacterial diet throughout infection and what other nutrients might be available at earlier time points.
Several lines of evidence indicated the igr
genes might be important for cholesterol metabolism. First of all, a homologous operon is present in Rhodococcus
strain RHA1 and is transcriptionally induced during growth on cholesterol (19
). In addition, a genome-wide genetic interaction screen predicted a functional association between the Mce4 transporter and products of the igr
). Finally, the predicted functions of several of these genes are consistent with a role in degrading either the side chain or sterol rings of the cholesterol molecule (19
In this work, we demonstrate that the H37Rv:Δigr mutant is unable to grow in the presence of cholesterol, although ATP generation and electron transport are unaffected. Thus, cholesterol addition appears to inhibit the growth of the mutant, likely via the accumulation of a stable catabolic intermediate. This hypothesis is supported by the ability of a mce4 mutation to reverse this toxicity, presumably by preventing the uptake of cholesterol. This effect is even more dramatic in vivo, where the Δmce4 mutation completely suppresses the Δigr-encoded phenotype. Detailed analysis of the replication rate of the Δigr mutant in vivo confirmed that this strain divides more slowly than the wild type during infection. However, in contrast to previous studies that monitored only CFU counts, we show here that the slow replication rate of this strain remains constant throughout infection and results in a reduced cumulative bacterial burden. Taken together, the results of this work demonstrate that the igr genes are essential for growth on cholesterol as a carbon source and that cholesterol is metabolized by M. tuberculosis throughout infection.