THE ROLE OF COMPLEMENT C3 AS A TRIGGER FOR HUMAN MACROPHAGE DEATH DURING INFECTION WITH FRANCISELLA TULARENSIS STRAIN SCHU S4

Abstract

Francisella tularensis, a Tier 1 Select Agent, is a highly infectious Gram-negative bacterium that causes the potentially deadly disease tularemia. Macrophages are considered a primary target of F. tularensis and serve as a replicative niche for the pathogen. F. tularensis escapes the phagosome to replicate in the host cell cytosol. Macrophage death is commonly observed following F. tularensis infections. The mechanism and significance of macrophage death in tularemia is not well understood, though the cause has been commonly attributed to the high bacterial burdens obtained following cytosolic replication. Research presented here indicates that extensive cytosolic replication of F. tularensis subsp. tularensis strain SCHU S4 in human monocyte-derived macrophages is neither necessary nor sufficient to induce macrophage death. Serum opsonization of F. tularensis results in iC3b deposition on the bacterial surface, which greatly enhances uptake by macrophages. Macrophage death was observed following uptake of F. tularensis in the presence of human serum, but not in the presence of C3-depleted serum, even when the multiplicities of infection were adjusted to ensure equal uptake under both conditions. This revealed that C3 plays an important role in macrophage death following infection with serum-opsonized F. tularensis. Single-cell analysis revealed that high bacterial burdens were not required for the induction of macrophage death. Infection with the mutant SCHU S4 ΔpurMCD, which can escape the phagosome but is deficient in cytosolic replication, resulted in macrophage death in a C3-dependent fashion like wild-type SCHU S4, supporting the conclusion that replication to high cytosolic burdens is not required for the induction of macrophage death. Infection with the mutant SCHU S4 ΔfevR, which cannot escape the phagosome but was taken up by macrophages at similar levels to wild-type bacteria in a C3-dependent manner, did not induce macrophage death. This revealed that engagement of complement receptors on the macrophage surface by uptake of iC3b-opsonized F. tularensis did not yield a sufficient signal to trigger macrophage death. One interpretation of these findings is that phagosome escape, but not extensive cytosolic replication of C3-opsonized F. tularensis is required for the induction of macrophage death. The existence of a cytosolic C3 sensor which leads to NF-B activation has been proposed by the same laboratory that identified TRIM 21 as a cytosolic antibody receptor. Here a hypothetical model is proposed in which C3 fragments enter the cytosol following phagosome escape of serum-opsonized F. tularensis, and that the response to cytosolic C3 in F. tularensis-infected macrophages is diverted to a cell death pathway resulting in host macrophage death.