Flow cytometry-based isolation combined with ultra-sensitive proteomics ofSalmonella-containing phagosomes reveals novel insights into host-pathogen interactions

Macrophages engulf pathogens into dynamic phagosomes, which many bacteria manipulate for survival. However, isolating pure pathogen-containing phagosomes remains challenging. Here, we developed a novel flow cytometry-based isolation and ultrasensitive proteomics approach to analyse phagosomal and bacterial proteomes from macrophages infected with wild-type (WT)Salmonella entericaserovar Typhimurium (STM), a ΔphoPmutant, or dead WT bacteria at 30 min and 4 hrs post-infection. Our approach provides higher throughput, requires lower cell numbers and quantifies more proteins than previous techniques. Our data reveals key host-pathogen interactions, showing induction of PhoP-dependent virulence factors and novel effectors to shape STM’s intracellular niche. Notably, phagosomes containing live WT STM resemble in some parts more those with dead bacteria than those with the PhoP mutant. Moreover, our data indicates that bacteria-containing phagosomes recruit mitochondrial membrane for production of reactive oxygen species. These findings provide new insights intoSalmonella’s manipulation of phagosomal maturation and intracellular niche formation.