Single-cell tracking of genetically minimized reveals mechanisms of effector gene cooperation.

Bacterial pathogens encode secretory systems that deliver large repertoires of effector proteins directly into host cells. While many effector proteins have been characterized biochemically, it is unclear where and when they function within complex cellular systems of host tissues. This problem exists because of the extensive genetic and functional redundancies found in large effector protein repertoires. Here, we coupled targeted genome minimization with single cell mass cytometry to track the cellular location where Typhimurium (Tm) SPI-2 Type Three Secretion System effector proteins function in a time-resolved animal model of infection. This approach revealed the temporal progression of Tm transmission within a complex tissue and pinpointed effector genes responsible for pathogen dissemination between host immune cell types. We further show how coupling two effector gene networks acquired during distinct episodes of bacterial evolution has shaped the cellular and tissue tropism of non-Typhoidal species. Together, these results illustrate a top-down genetic approach to interrogate host-pathogen interactions hidden by functional redundancies within large virulence factor gene networks.