Flow cytometry-based isolation combined with ultra-sensitive proteomics of Salmonella-containing phagosomes 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.; Heunis, T.; Li, M.; Filby, A.; Trost, M.

doi:10.1101/2025.04.25.650444

bioRxiv · 10.1101/2025.04.25.650444

Flow cytometry-based isolation combined with ultra-sensitive proteomics of Salmonella-containing phagosomes 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.·Heunis, T.·Li, M.·Filby, A.·Trost, M.

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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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Chatterjee, R., Marin-Rubio, J. L., Cianfanelli, F. R., Frey, A. M., Raymond, B. B. A., Meng, M., Dannoura, A., Heunis, T., Li, M., Filby, A., Trost, M.. 2025-04-25. Flow cytometry-based isolation combined with ultra-sensitive proteomics of Salmonella-containing phagosomes reveals novel insights into host-pathogen interactions. https://doi.org/10.1101/2025.04.25.650444

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