Flow cytometry-based isolation and ultra-sensitive proteomics of Salmonella-containing phagosomes provides novel insights into pathogen interactions with host cells

ABSTRACT: Bacterial infections can be life-threatening to both humans and animals, an affliction bearing a tremendous economic burden-as testified by the incidence of antimicrobial resistance. During infection, innate immune cells from the host present the first line of defense as invading pathogens are engulfed into specialized de novo organelles called phagosomes. These phagosomes mature into lysosomes, establishing a hostile environment in which the pathogen is effectively neutralized and degraded. Most intracellular pathogens actively manipulate the maturation of phagosomes as a means to support their survival and replication within immune cells like macrophages, such as Salmonella enterica spp. Although this interaction is of paramount importance, the details about its molecular level are not well understood because of a number of technical difficulties in the isolation of pure bacteria-containing phagosomes and their highly complex network. Furthermore, even within one macrophage, phagosomes exhibit extreme heterogeneity, which pathogens exploit for their own benefit. In the face of these challenges, we have developed a novel approach using antibody-free fluorescence-activated cell sorting to purify intact bacteria-containing phagosomes. Subsequent ultrasensitive mass spectrometry-based proteomics thus allow the identification of host and bacterial proteins present on these phagosomes at nano-gram levels. We applied this strategy for the purification of Salmonella-containing vacuoles (SCVs) from infected human THP-1 macrophages highlighting an tug-of-war kind of host-pathogen battle. Our results show reproducibility and robustness of our strategy and confirm literature while revealing new information on host proteins playing a role in magnesium transport across the SCV membrane at various steps of infection. Moreover, we observed changes in the host and bacterial proteins, in terms of stress, innate immunity, and virulence responses, are induced through what the Salmonella PhoP/Q system, one of the most basic influences on the human phagosome proteome in a time-dependent manner, has provided.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bacteria Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. Sl1344

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Matthias Trost  

LAB HEAD: Matthias Trost

PROVIDER: PXD062621 | Pride | 2026-07-28

REPOSITORIES: Pride