Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence.
Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence.
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DOI：
10.1371/journal.ppat.1006363
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发表时间：
2017-05
期刊：
影响因子：
6.7
通讯作者：
Hung DT
中科院分区：
文献类型：
--
作者：
Barczak AK;Avraham R;Singh S;Luo SS;Zhang WR;Bray MA;Hinman AE;Thompson M;Nietupski RM;Golas A;Montgomery P;Fitzgerald M;Smith RS;White DW;Tischler AD;Carpenter AE;Hung DT
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. Tuberculosis (TB) remains a significant global health problem. One barrier to developing novel approaches to preventing and treating TB is an incomplete understanding of the strategies that the causative bacterium, Mycobacterium tuberculosis (Mtb), uses to survive and cause disease in the host. 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. We then used multiplexed cytokine analysis to profile the macrophage response to each mutant attenuated for intracellular growth. 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. We then validated these predictions by demonstrating that production and export of a cell envelope lipid is required for coordinated virulence-associated protein secretion, phagosomal membrane rupture, and production of the macrophage type I interferon response. Extending our prediction of functional relationships to unknown genes, we demonstrated that two genes not previously linked to virulence also act in this pathway. This work demonstrates a broadly applicable approach to elucidating and relating bacterial functions required for pathogenesis and demonstrates a previously unknown dependence of Mtb virulence-associated protein secretion on an outer envelope lipid.
