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Researchers developed PhagoCyt, a new flow cytometry-based method combined with ultrasensitive proteomics to isolate and analyze phagosomes containing Salmonella bacteria from infected macrophages. The technique requires fewer cells and identifies more proteins than previous methods, revealing how Salmonella manipulates its cellular environment through PhoP-dependent virulence factors and how infected phagosomes recruit mitochondrial membranes to produce reactive oxygen species. The study compared wild-type Salmonella with a mutant strain lacking the PhoP gene at two time points after infection to map dynamic changes in both host and bacterial proteins.
Why it matters
This improved methodology enables more detailed study of how intracellular pathogens manipulate host cells to survive, which could inform development of new antimicrobial strategies targeting these host-pathogen interactions. The identification of novel proteins involved in Salmonella's intracellular survival provides potential therapeutic targets for combating antibiotic-resistant infections.
Understand the Science
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
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 ΔphoP 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 Salmonella’s manipulation of phagosomal maturation and intracellular niche formation.