Innate immune cells detect, engulf, and eliminate pathogens through multiple reactions including acidification and proteolytic activity inside intracellular organelles called phagosomes. Although previous studies have monitored individual reactions in phagosome “maturation” process, the temporal correlation between acidification and proteolytic activity of phagosomes has not been studied. A challenge to address this question is to monitor multiple reactions inside single phagosomes simultaneously. We have tackled this challenge and developed a bifunctional Janus particle probe for the simultaneous monitoring of acidification and proteolysis within single phagosomes in living macrophage cells. Each probe consists of fluorescence-based a pH reporter and a proteolysis reporter that are spatially separated but function concurrently. Using the bifunctional probes, we measured intracellular pH and proteolytic activity in single phagosomes. We found that the acidic environment is a prerequisite to initiate and maintain proteolytic activities, demonstrating the sequential occurrence of both reactions during phagosome maturation and providing a quantitative explanation. Our results further identified that phagosome maturation is altered by the presence of bacterium-derived lipopolysaccharide.

Simultaneous real-time imaging of acidification and proteolysis inside single phagosomes

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