Salmonella multimutants enable efficient identification of SPI-2 effector protein function in gut inflammation and systemic colonization

Salmonella enterica relies on translocation of effector proteins through the SPI-2 type III secretion system (T3SS) for pathogenesis. More than 30 effectors contribute to manipulation of host cells through diverse mechanisms, but interdependency or redundancy between effectors complicates the discovery of effector phenotypes using single mutant strains. Here, we engineer six mutant strains to be deficient in groups of SPI-2 effectors, as defined by their reported function. Using various animal models of infection, we show that three main phenotypes define the functional contribution of the SPI-2 T3SS to infection. Multimutant strains deficient for intracellular replication, for manipulation of host cell defences, or for expression of virulence plasmid effectors all show strong attenuation in vivo, while mutants representing approximately half of the known effector complement show phenotypes similar to the wild-type parent strain. By additionally removing the SPI-1 T3SS, we find groups of effectors that contribute to SPI-2 T3SS-driven enhancement of gut inflammation. Further, we provide an example of how iterative mutation can be used to find a minimal number of effector deletions required for attenuation, and thus establish that the SPI-2 effectors SopD2 and GtgE are crucial for promotion of gut inflammation and mucosal pathology.