Abstract
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.
Highlights
A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the capacity to survive within host macrophages
To systematically identify Mtb genes required for growth in infected host cells, we screened an annotated, arrayed library of Mtb mutants in macrophages using high-content imaging
Combining imaging parameters reflective of intracellular infection with the macrophage response to each mutant, we predicted novel functional relationships between Mtb genes required for infection
Summary
A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the capacity to survive within host macrophages. Several screening approaches have identified Mtb factors required for its intracellular survival. Using large mixed pools of Mtb mutants, Pethe et al identified mutants that co-localized with iron-containing lysosomes [2]. Using highcontent imaging to screen a library of 11,000 randomly selected, arrayed transposon mutants, Brodin et al identified 10 mutants unable to block phagosome maturation based on co-localization with LysoTracker-stained acidified lysosomes [3]. An alternative genomic approach focused on Mtb growth in macrophages and compared input and output pools of transposon mutants to identify several attenuated mutants [4]. There is little overlap between the sets of Mtb genes identified as important in macrophages using each of these approaches. Of the 10 genes identified by Brodin et al, only one (pstS3) was identified in the screen by Rengarajan et al, and only one (fadD28) was identified by Pethe et al There is no overlap between genes identified by Rengarajan et al and those identified by Pethe et al
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