<p>by Ritika Chatterjee, José Luis Marin-Rubio, Francesca Romana Cianfanelli, Andrew Michael Frey, Benjamin Bernard Armando Raymond, Abeer Dannoura, Camila Valenzuela, Meihan Meng, Tiaan Heunis, Mengchun Li, Frances Sidgwick, Jost Enninga, Andrew Filby, Matthias Trost</p> 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) <i>Salmonella enterica</i> serovar Typhimurium (STM) or a <i>ΔphoP</i> mutant at 30 min and 4 hours 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 putative proteins that shape STM’s intracellular niche. Moreover, our data indicates that bacteria-containing phagosomes recruit mitochondrial membrane for production of reactive oxygen species. These findings provide new insights into <i>Salmonella</i>’s manipulation of phagosomal maturation and intracellular niche formation.