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

Chatterjee, R.; Marin-Rubio, J. L.; Cianfanelli, F. R.; Frey, A. M.; Raymond, B. B. A.; Meng, M.; Dannoura, A.; Valenzuela-Montenegro, C.; Heunis, T.; Li, M.; Enninga, J.; Filby, A.; Trost, M.

2025-10-03 microbiology
10.1101/2025.04.25.650444 bioRxiv
Show abstract

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 enterica serovar Typhimurium (STM) or a {Delta}phoP mutant 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 putative proteins that shape STMs 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 Salmonellas manipulation of phagosomal maturation and intracellular niche formation.

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