Syk Plays an Essential Role in Phagosome-Lysosome Fusion By Facilitating Actin-Remodeling in Complement-Mediated Phagocytosis

Complement-mediated phagocytosis plays an essential role in host defense against invading pathogens. Phagocytosis requires a dynamic and coordinated reconstruction of the membrane and the underlying cytoskeleton. Pathogens are entrapped into a phagosome and internalized into the cell. Subsequently, the phagosome fuses with lysosomes to form a phagolysosome with gradual acidification. Previous studies showed the pivotal roles of phosphoinositide-mediated signaling and the synchronous actin-remodeling in phagosome formation, but the mechanisms of phagolysosome generation, namely phagosome-lysosome fusion remain largely unexplored.

Here we show that phagosome-lysosome fusion requires the collapse of F-actin structure surrounding phagosomes and a tyrosine kinase, Syk plays a key role in this process. To reveal the mechanisms of phagosome-lysosome fusion particularly focusing on Syk, we performed complement-mediated phagocytosis assay using human leukemic HL60 and Syk-knockout (Syk-KO) HL60 cells established by the CRISPR/Cas9 system. Parental HL60 and Syk-KO HL60 were differentiated into macrophage-like cells and incubated with complement-opsonized Candida albicans(C. albicans) or fluorescence-labeled zymosan particles.

Syk-KO cells permitted the survival of C. albicansand their escape from the phagosome.In general, after fusion with lysosomes, the phagosome acquires acidic milieu inside the organelle and enhanced bactericidal activity. To confirm the effects of Syk-KO on phagosome acidification, we used two types of fluorescence-labeled zymosan particles: one was labeled with fluorescein isothiocyanate (FITC) whose fluorescent intensity is reduced under decreased pH condition and the other was labeled with Texas Red whose intensity is conserved independent of pH change. Flow cytometric analysis indicated that Syk-KO cells showed insufficient phagosome acidification. These results suggest that Syk facilitates acidification of phagosomes and results in an enhanced bactericidal activity against pathogens following complement-mediated phagocytosis.

Since ourprevious study indicated the participation of Syk in dynamics of actin cytoskeleton, we examined the effect of Syk-KO on the distribution of F-actin around phagosomes. Two hours after phagocytosis, pathogen-containing phagosomes of both parental and Syk-KO HL60 cells were already intracellularly distributed. At that time, F-actin structure around phagosomes of parental HL60 cells was hardly invisible but the phagosomes of Syk-KO cells were circumscribed by dense F-actin structure. An F-actin-stabilizing agent, jasplakinolide induced a similar fusion defect in peripheral blood-derived monocytes as observed in Syk-KO cells. Presence of dense F-actin structure surrounding phagosomes seemed to be responsible for inhibiting phagosome-lysosome fusion. In other words, collapse of F-actin structure surrounding phagosomes may be a critical step of phagolysosome generation.

Our results demonstrate that actin-remodeling is essential not only for phagosome formation but also for phagosome-lysosome fusion in complement-mediated phagocytosis and that Syk promotes this process. The present study provides new insights into the mechanism controlling phagocytic progression that leads to host defense against pathogens.