Chapter 35 : The Sculpting of the Mycobacterium tuberculosis Genome by Host Cell–Derived Pressures

Genetic and transcriptome diversity of M. tuberculosis complex (MTC) clinical isolates. (A) Radial neighbor-joining tree based on 24 loci MIRU-VNTR and 43 spacer spoligotyping showing the phylogenetic relationship of strains in this study. Three strains each from the five distinct lineages pathogenic to humans plus two sequenced reference strains (H37Rv and CDC1551) were chosen to represent the global diversity of MTC. (B) Condition tree of MTC clinical isolate transcription profiles in vitro during log phase growth in 7H9 medium relative to the CDC1551 reference strain (three biological replicates). Expression profiles for genes passing quality filters (flagged as present in 42 of 48 samples) with differential expression in at least one strain (up or down >1.5-fold in at least 1 of 16) were clustered using the Spearman correlation. Each column represents the global transcription profile of a single strain. Genes were clustered vertically based on the distance measure. Red and blue indicate higher or lower gene expression than the CDC1551 control, respectively. This figure is reproduced from Homolka et al. ( 28 ). doi:10.1128/microbiolspec.MGM2-0016-2013.f1

Life and death dynamics during long-term intracellular survival of M. tuberculosis. (A) Survival assays. Resting murine bone-marrow-derived macrophages were infected at low multiplicity of infection (∼1:1) with M. tuberculosis CDC1551. Viable CFUs were quantified at day 0 and at 2-day intervals postinfection over a 14-day time course by lysis of monolayers, serial dilution, and plating on 7H10 medium. Error bars indicate standard error of the mean from two independent biological replicates, each consisting of three technical replicates per strain (total of six wells/strain). (B) Replication clock plasmid. The percentage of bacteria containing the pBP10 plasmid during growth in resting macrophages was determined by comparing CFUs (mean ± standard deviation) recovered on kanamycin vs. nonselective media (red). The cumulative bacterial burden (CBB) (black) was determined by mathematical modeling based on total viable CFUs and plasmid frequency data. Data shown represent two independent experiments, with each sample performed in quadruplicate (eight total wells/time point). (C) The “bottleneck” response. Temporal expression profiles of genes differentially regulated at day 2 postinfection, including genes from (A) that were up- (red) or downregulated (blue) >1.5-fold (shown as ratio of signal intensity relative to control). Note the maximal change in transcript levels at day 2 postinfection followed by the majority trending back toward control levels. (D) “Guilt by association” analysis. Genes regulated in synch with known virulence regulons (i.e., the DosR regulon) were identified by using a highly regulated member of this regulon, hspX, in place of synthetic profiles. This figure is reproduced from Rohde et al. ( 29 ). doi:10.1128/microbiolspec.MGM2-0016-2013.f2

Distribution of zinc within intraphagosomal Mycobacterium avium. Bone-marrow-derived mouse macrophages were infected with M. avium and treated for capturing of free zinc ions by an auto-metallography (AMG) procedure. Cells were then processed for electron microscopy observation. The zinc crystals formed during this procedure appeared as small dense deposits along the inner face of the phagosome membrane (arrows). The mycobacterial cytoplasm was devoid of zinc. Lysosomes also displayed zinc crystals. Comparable images were observed in M. tuberculosis–infected macrophages. Scale bar, 0.5 µm. This picture was provided by Dr. Chantal de Chastellier and was prepared as detailed in reference 74 . doi:10.1128/microbiolspec.MGM2-0016-2013.f3

Immunoelectronmicroscopy of M. tuberculosis–infected macrophages. All sections were probed with mouse antiubiquitin (12 nm gold) and rat anti-LAMP1 (6 nm gold). (A) Untreated, infected macrophage demonstrating that the bacteria-containing vacuoles lack ubiquitin signal and that the ubiquitin signal is predominantly associated with either the membranes (horizontal arrows) or the lumen (vertical arrowheads) of LAMP1-positive, dense lysosomes. (B) In cells treated by starvation for 2 h, the M. tuberculosis–containing vacuole has acquired a dense, LAMP1-positive, flocculent matrix that is also positive for ubiquitin (arrowed), demonstrating delivery of lysosomal ubiquitin to the bacteria-containing compartment. The relative distribution of ubiquitin-associated label in M. tuberculosis–containing vacuoles in untreated and starved bone marrow-derived macrophages is detailed in Fig. 4D, with standard error bars. This figure is reproduced from Alonso et al. ( 77 ). doi:10.1128/microbiolspec.MGM2-0016-2013.f4

Peripilin 2/ADFP expression in human tuberculosis granulomas. Immunofluorescence signals were obtained for each granuloma (B), and the corresponding region from a hematoxylin and eosin stained slide (A) is shown. Nuclei are shown in blue and antigens in red. The macrophages subtending the caseum of this fibrocaseous granuloma label strongly for peripilin2/ADFP expression. This figure is reproduced from Kim et al. ( 88 ). doi:10.1128/microbiolspec.MGM2-0016-2013.f5

Outline of the pathways relevant to C3 metabolism. The metabolism of cholesterol, methyl-branched fatty acids, and odd-chain length lipids raises the intracellular levels of the C3 compounds propionate or propionyl-CoA, which M. tuberculosis finds highly toxic. The bacterium has developed three strategies to detoxify propionyl-CoA. Isocitrate lyase (icl1) activity has been suborned to fulfill the function of methylcitrate lyase in the last step of the methyl citrate cycle to generate the TCA cycle intermediate succinate. The methylmalonyl pathway has also been mobilized to metabolize propionyl-CoA to produce succinyl-CoA via the vitamin B12-dependent activity of methylmalonyl-CoA mutase (mutAB). Finally, intermediates from the methylmalonyl pathway can be incorporated directly into the abundant, methyl-branched lipids of the bacterial cell wall, such as PDIM and SL-1. doi:10.1128/microbiolspec.MGM2-0016-2013.f6

Intracellular growth is restored to the Δicl1 M. tuberculosis through the induction of oleate-containing lipid droplets in the infected cell. The survival of the Δicl1 mutant is impaired in macrophages, but growth could be restored by preloading the host cells with exogenous oleate or by adding vitamin B12 to the cell culture medium to facilitate operation of the methyl malonyl pathway. (Upper) Induction of macrophage lipid droplets, detected with BODIPY 493/503 (green), M. tuberculosis expressing pVV16-mCherry (smyc′::mCherry) (red), and nuclei (blue) in oleate-loaded macrophages versus untreated macrophages. In the absence of M. tuberculosis infection, the lipid droplets are lost from the macrophages in 48 h. However, in the presence of M. tuberculosis, the lipid droplets persist. (Lower) Bacterial CFUs were determined at 2-day intervals across an 8-day period for the Δicl1 M. tuberculosis in untreated, control macrophages (triangle), in lipid droplet–containing, oleate-loaded macrophages (square), and in macrophages supplemented by the addition of vitamin B12 to the medium (diamond). Error bars indicate the standard error of the mean from three representative replicates. This figure is reproduced from Podinovskaia et al. ( 90 ) and Lee et al. ( 102 ). doi:10.1128/microbiolspec.MGM2-0016-2013.f7