Cross-membrane cooperation among bacteria can facilitate intracellular pathogenesis.

is a Gram-negative opportunistic pathogen able to cause life- and sight-threating infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among subpopulations can contribute to disseminating intracellular bacteria in a mouse infection model. This study provides the basis for future studies to investigate how cooperation of extracellular and intracellular bacteria within the host may contribute to disease progression and persistence.