A Genetic Screen forMycobacterium tuberculosisMutants Defective for Phagosome Maturation Arrest Identifies Components of the ESX-1 Secretion System

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Abstract
After phagocytosis, the intracellular pathogenMycobacterium tuberculosisarrests the progression of the nascent phagosome into a phagolysosome, allowing for replication in a compartment that resembles early endosomes. To better understand the molecular mechanisms that govern phagosome maturation arrest, we performed a visual screen on a set ofM. tuberculosismutants specifically attenuated for growth in mice to identify strains that failed to arrest phagosome maturation and trafficked to late phagosomal compartments. We identified 10 such mutants that could be partitioned into two classes based on the kinetics of trafficking. Importantly, four of these mutants harbor mutations in genes that encode components of the ESX-1 secretion system, a pathway critical forM. tuberculosisvirulence. Although ESX-1 is required, the known ESX-1 secreted proteins are dispensable for phagosome maturation arrest, suggesting that a novel effector required for phagosome maturation arrest is secreted by ESX-1. Other mutants identified in this screen had mutations in genes involved in lipid synthesis and secretion and in molybdopterin biosynthesis, as well as in genes with unknown functions. Most of these trafficking mutants exhibited a corresponding growth defect during macrophage infection, but two mutants grew like wild-typeM. tuberculosisduring macrophage infection. Our results support the emerging consensus that multiple factors fromM. tuberculosis, including the ESX-1 secretion system, are involved in modulating trafficking within the host.