Type III secretion system (T3SS) effectors are key virulence factors that underpin the infection strategy of many clinically important Gram-negative pathogens, including Salmonella enterica, Shigella spp, enteropathogenic and enterohaemorrhagic Escherichia coli and their murine equivalent, Citrobacter rodentium. The cellular processes or proteins targeted by the effectors can be common to multiple pathogens or pathogen-specific. The main approach to understanding T3SS-mediated pathogenesis has been to determine the contribution of one effector at a time, with the aim to piece together individual functions and unveil infection mechanisms. However, in contrast to this prevailing approach, simultaneous deletion of multiple effectors revealed that they function as an interconnected network in vivo, uncovering
effector co-dependency and context-dependent effector essentiality. This paradigm shift in T3SS biology is at the heart of this opinion.