Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages

Abstract

Macrophages are central to host defense against microbes, but intracellular pathogens have evolved to evade their antimicrobial functions. Mycobacterium tuberculosis (MTB) has successfully exploited macrophages as its primary niche in vivo, but the bacterial genome-wide requirements that promote its intracellular survival remain undefined. Here we comprehensively identify the MTB genes required for survival by screening for transposon mutants that fail to grow within primary macrophages. We identify mutants showing decreased growth in macrophage environments that model stages of the host immune response. By systematically analyzing several biologically relevant data sets, we have been able to identify putative pathways that could not be predicted by genome organization alone. In one example, phosphate transport, requiring physically unlinked genes, was found to be critical for MTB growth in macrophages and important for establishing persistent infection in lungs. Remarkably, the majority of MTB genes found by this analysis to be required for survival are constitutively expressed rather than regulated by macrophages, revealing the host-adapted lifestyle of an evolutionarily selected intracellular pathogen.

Fig. 1.

Comprehensive screen identifies genes required for surviving within macrophages under different activation conditions. (a) TraSH in primary murine macrophages. We infected pools of macrophages derived from bone marrows of C57BL/6 mice with 10⁷ MTB mariner transposon mutants, moi of 1:1. To identify MTB genes required for survival within macrophages, we lysed macrophages after 7 days and plated surviving bacteria. Mutants that failed to survive this macrophage selection (macrophage pool) are absent in the recovered bacteria. These mutants are present when the library is directly plated (in vitro pool). To map transposon insertion sites in each pool, genomic DNA from the macrophage and in vitro pools was isolated and TraSH probe generated that was complementary to the chromosomal sequence flanking insertion sites in each pool. We compared mutants from the macrophage pool to the in vitro pool by labeling the probe from each pool with a different colored fluorophore, mixing and hybridizing them to a spotted MTB genome microarray. Spots that hybridized to the probe from the in vitro pool but not the macrophage pool correspond to genes required for growth in macrophages. To enrich for such mutants, we reinfected a fresh pool of macrophages with bacteria that survived the first round of infection and performed TraSH. Data from the enrichment were used in subsequent analyses. (b) Genes required for survival in macrophages. A vertical line corresponds to each experimental condition; macrophages that are unactivated or activated with IFN-γ pre- or postinfection. The y axis corresponds to average ratios of in macrophage/in vitro growth, and mutants with ratios <0.4 or >4 (P < 0.05) are represented. Each line indicates the growth of the mutant in macrophages/in vitro across the three conditions of macrophage culture. Values <1.0 (blue) represent mutants that grow poorly in macrophages, whereas ratios >1.0 (red) are mutants that show enhanced intracellular growth. (c) k means clustering of MTB genes required for survival in any of the three macrophage conditions. Genes with ratios differing from the median by 2.5-fold or less (P < 0.05) were clustered into 10 clusters and represented in a 3D scatter plot when each axis corresponds to the indicated condition. Each color corresponds to a different cluster.

Fig. 2.

Hierarchical clustering of genes required for survival in macrophages (unactivated, IFN-γ preinfection, and IFN-γ postinfection) and in vivo in mouse spleens (1, 2, 4, and 8 weeks postinfection). Blue indicates decreased survival of mutant in macrophages relative to growth in vitro. Red indicates increased survival of mutant in macrophages relative to in vitro

Fig. 3.

pstA1 and phoT, components of a phosphate transport apparatus, are essential for MTB growth in phosphate starved conditions in vitro (a), in macrophages (unactivated or activated with IFN-γ preinfection or IFN-γ postinfection) (b), and phoT is required for growth in lungs of mice (c).

Fig. 4.

Hierarchical clustering of genes required for survival in macrophages (lane 1, unactivated; lane 2, IFN-γ preinfection; lane 3, IFN-γ postinfection) with genes induced in infected wild-type (C57BL/6) macrophages 4, 24, and 48 h after infection in unactivated (lane 6, 24 h; lane 7, 48 h; lane 9, 4 h) or pre-IFN-γ-activated macrophages (lane 8, 4 h; lane 10, 24 h; lane 11, 48 h) or in NOS2-/- macrophages unactivated (lane 4, 24 h) or pre-IFN-γ-activated (lane 5, 24 h). Blue indicates decreased survival of mutant in macrophages relative to in vitro (survival data) or down-regulation of genes after infection of macrophages, relative to expression in vitro (expression data). Red indicates increased survival of mutant in macrophages relative to in vitro or up-regulation of genes after infection in macrophages. Shades of color correspond to numerical ratios and are indicated by the color key.