The Gram-negative bacterial type VI secretion system (T6SS) delivers toxins to kill or inhibit the growth of susceptible bacteria, while other secretion systems target eukaryotic cells. Deletion of , a negative regulator of virulence factors in K56-2, increases T6SS activity. Macrophages infected with a K56-2 mutant display dramatic alterations in their actin cytoskeleton architecture that rely on the T6SS, which is responsible for the inactivation of multiple Rho-family GTPases by an unknown mechanism. We employed a strategy to standardize the bacterial infection of macrophages and densitometrically quantify the T6SS-associated cellular phenotype, which allowed us to characterize the phenotype of systematic deletions of each gene within the T6SS cluster and ten genes in K56-2 . None of the genes from the T6SS core cluster nor the individual genes were directly responsible for the cytoskeletal changes in infected cells. However, a mutant strain with all genes deleted was unable to cause macrophage alterations. Despite not being able to identify a specific effector protein responsible for the cytoskeletal defects in macrophages, our strategy resulted in the identification of the critical core components and accessory proteins of the T6SS assembly machinery and provides a screening method to detect T6SS effectors targeting the actin cytoskeleton in macrophages by random mutagenesis.


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