The Shumin Tan Lab
Environmental Cues in Mycobacterium tuberculosis-Host Interactions
Mycobacterium tuberculosis (Mtb) is an extremely successful pathogen that remains a key public health problem, with broad impact on population and economic development. We seek to understand the molecular mechanisms of Mtb-host interactions, with a focus on the host environmental signals that serve as cues for Mtb during host colonization. Knowledge of how environmental cues in vivo are integrated with core aspects of Mtb biology is fundamental to our understanding of Mtb infection and tuberculosis disease progression.
We have discovered that chloride concentration is inversely related to pH during phagosomal maturation. Using a chloride and pH-responsive fluorescent reporter Mtb strain, we found that Mtb responds synergistically to these two cues, with the response reflecting the host’s immune status (Figure 1). Building on this, we are studying how Mtb may link its response to various environmental signals, and exploring the novel concept that local concentrations of abundant ions can profoundly impact a pathogen’s ability to colonize its host. Elucidation of nodes critical to the bacterium’s ability to sense and respond to environmental cues open up potential avenues for targeting Mtb to shift the balance of infection in favor of the host.
Figure 1. (A) Mtb responds synergistically to chloride and pH, as illustrated by differential induction of GFP signal in reporter Mtb (rv2390c’::GFP) engineered to express GFP in response to increased chloride concentration or decreased pH in its environment. (B) In a murine infection model, this reporter signal (green) is higher in the bacteria present in lungs of wild type (WT) versus immune-deficient interferon gamma (IFNγ) knockout hosts 28 days post-infection. All Mtb were also engineered to constitutively express mCherry (smyc’::mCherry, red) for visualization of the bacteria in the lung tissue. Host nuclei are in grayscale, and phalloidin staining of f-actin is shown in blue in these 3D confocal images.
Mycobacterium tuberculosis Heterogeneity during Infection
A hallmark of Mtb infection is the marked heterogeneity present. Infection foci in the host progress differently and independently of one another, with consequent impact on treatment efficacy. Our in vivo studies utilizing reporter Mtb strains have served to illustrate the extent of this heterogeneity – for example, heterogeneity in the environmental signals experienced by the bacteria during infection extends to the level of individual infection foci and host cells (Figures 2A and 2B). We are pursuing studies to understand how heterogeneity in environmental cues impact on Mtb colonization, and have developed a replication reporter Mtb strain (SSB-GFP) to aid in these studies, where active DNA replication in the bacteria is marked by the presence of green foci (Figure 2C). Reporter Mtb strains allow us to examine questions of heterogeneity at the individual bacterium level, while maintaining spatial context and the full complexity of in vivo host infection. By exploiting these strains in infection models and combining it with thick tissue imaging and 3D confocal reconstruction, we aim to elucidate how environmental signals may drive heterogeneity, and factors that distinguish an infection focus as conducive or restrictive for Mtb growth in vivo.
Figure 2. (A and B) 3D confocal images of lung tissue from a 28 and 14 day murine infection respectively, illustrating the heterogeneity observed during Mtb host infection. Reporter signal is in green (rv2390c’::GFP), all Mtb are in red (smyc’::mCherry), nuclei are in grayscale (DAPI), and phalloidin staining of f-actin is in blue where shown. (C) SSB-GFP replication reporter marks active Mtb DNA replication with green foci. The bacteria also constitutively express mCherry via a smyc’::mCherry construct (red).