Person:

Hung, Deborah

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the capacity to survive within host macrophages. Although several factors required for this survival have been identified, a comprehensive knowledge of such factors and how they work together to manipulate the host environment to benefit bacterial survival are not well understood. To systematically identify Mtb factors required for intracellular growth, we screened an arrayed, non-redundant Mtb transposon mutant library by high-content imaging to characterize the mutant-macrophage interaction. Based on a combination of imaging features, we identified mutants impaired for intracellular survival. We then characterized the phenotype of infection with each mutant by profiling the induced macrophage cytokine response. Taking a systems-level approach to understanding the biology of identified mutants, we performed a multiparametric analysis combining pathogen and host phenotypes to predict functional relationships between mutants based on clustering. Strikingly, mutants defective in two well-known virulence factors, the ESX-1 protein secretion system and the virulence lipid phthiocerol dimycocerosate (PDIM), clustered together. Building upon the shared phenotype of loss of the macrophage type I interferon (IFN) response to infection, we found that PDIM production and export are required for coordinated secretion of ESX-1-substrates, for phagosomal permeabilization, and for downstream induction of the type I IFN response. Multiparametric clustering also identified two novel genes that are required for PDIM production and induction of the type I IFN response. Thus, multiparametric analysis combining host and pathogen infection phenotypes can be used to identify novel functional relationships between genes that play a role in infection.