Phenotypic Heterogeneity in Mycobacterium tuberculosis

Causes and consequences of phenotypic heterogeneity. Bacterial isogenic populations arising from a single progenitor cell are usually expected to be homogeneous (left snapshot). However, single-cell analysis unveils significant cell-to-cell heterogeneity (right snapshot). Some of the causal factors leading to this heterogeneity are variations in growth rate, growth continuity, interdivision time, division symmetry, gene expression, protein distribution, and cell age generated either through deterministic or stochastic mechanisms.

Stress conditions enhance M. tuberculosis phenotypic heterogeneity. Upper panel, single-cell rRNA-GFP fluorescence of M. tuberculosis isolated from different environments: Exp (exponential phase), Stat (stationary phase), Drug (treated with isoniazid), Mɸ (grown in macrophages), and Mouse (explanted from mouse lungs during the acute phase of infection). Each circle represents a single cell and the mean fluorescence ± SD is indicated (n = 200 per time point). Asterisks indicate significance difference of each data set in comparison with the control group, Exp (P < 0.0001), according to ANOVA followed by the Kruskal-Wallis test. The numbers shown on top are the coefficient of variation (CV) for each data set. Lower panel, representative snapshots from the corresponding conditions are shown. Green (rRNA-GFP) and red (constitutive dsRed) fluorescence channels are merged. Macrophages are also shown in phase contrast. Scale bars, 5 μm. Figure adapted from Manina et al. ( 35 ).

Identification of NGMA bacteria by single-cell techniques. A schematic of the fluorescence recovery after photobleaching (FRAP) method is shown on the top. M. tuberculosis expressing cytoplasmic rRNA-GFP were subjected to photobleaching using a laser, followed by staining with a dye that penetrates only cells with a compromised membrane. Metabolically active cells (green), bleached or metabolically inactive cells (gray), and dead cells (blue) are depicted. Representative snapshots of stationary-phase M. tuberculosis cells that were exposed to fresh 7H9 medium for 1 week. Top row, a nongrowing cell that does not recover fluorescence after photobleaching and stains positive for dead-cell stain (negative control). Middle row, a nongrowing cell that recovers fluorescence after photobleaching and stains negative for dead-cell stain and is therefore identified as nongrowing but metabolically active (NGMA). Bottom row, a growing cell that recovers fluorescence after photobleaching, stains negative for dead-cell stain, and continues to grow postbleaching.

Host and pathogen heterogeneity contributes to TB diversity. Schematic of the increasingly heterogeneous environments M. tuberculosis resides in. Disease heterogeneity initiates in the major site of infection where host immunity gives rise to the typical granulomatous lesion (magnified from the lung parenchyma). This assembly of host cells consists of different types of macrophages, dendritic cells, neutrophils, lymphocytes, fibroblasts, and a necrotic caseous core. Bacilli can reside in discrete niches both intracellularly (magnified from the granuloma) and extracellularly, where they are subjected to a plethora of host immune effectors (red arrow) and antibiotics (blue arrow). Diverse environmental cues found within each microniche contribute to enhance the intrinsic phenotypic diversity of M. tuberculosis (right snapshot).

Cellular dynamic processes and single-cell techniques. The different biological processes that occur during cell growth and that often determine cell fate and some of the techniques that are used to track these processes at the single-cell level are depicted. Fluorescent approaches involve the use of fluorescent protein fusions or fluorescent-tagged molecules. Figure adapted from Spiller et al. ( 227 ).