Vacuolar and plasma membrane stripping and autophagic elimination of Toxoplasma gondii in primed effector macrophages

Abstract

Apicomplexan protozoan pathogens avoid destruction and establish a replicative niche within host cells by forming a nonfusogenic parasitophorous vacuole (PV). Here we present evidence for lysosome-mediated degradation of Toxoplasma gondii after invasion of macrophages activated in vivo. Pathogen elimination was dependent on the interferon gamma inducible-p47 GTPase, IGTP, required PI3K activity, and was preceded by PV membrane indentation, vesiculation, disruption, and, surprisingly, stripping of the parasite plasma membrane. Denuded parasites were enveloped in autophagosome-like vacuoles, which ultimately fused with lysosomes. These observations outline a series of mechanisms used by effector cells to redirect the fate of a classically nonfusogenic intracellular pathogen toward a path of immune elimination.

Figure 1.

IGTP-dependent rapid T. gondii elimination in primed macrophages. (A) Impaired T. gondii elimination in primed IGTP^−/− mice. CPS-primed (day 7 after infection) WT and IGTP^−/− mice were challenged with 4 × 10⁶ GFP-PTG i.p. Infection rates (mean ± SEM) in peritoneal macrophages were determined by FACS (n = 3). (B) Survival of primed WT (n = 6) and IGTP^−/− (n = 4) mice after GFP-PTG challenge. (C) Infection rates in primed WT and IGTP^−/− peritoneal macrophages after GFP-PTG infection (MOI of 1, 15 min) ex vivo. Data shown are representative of eight experiments. (D) Thioglycolate-elicited WT and IGTP^−/− peritoneal macrophages failed to eliminate T. gondii after GFP-PTG infection (MOI of 1, 15 min) when activated in vitro. IFN-γ treatment (1 ng/ml) started 12 h before infection. (E) IGTP-dependent rapid T. gondii elimination in primed macrophages does not require iNOS. Primed (WT, IGTP^−/−, iNOS^−/−, and IGTP^−/−iNOS^−/−) and naive WT (thioglycolate-elicited) PECs were infected with GFP-PTG ex vivo. Data are representative of three experiments.

Figure 2.

Evidence for lysosomal fusion with T. gondii in primed macrophages. (A) T. gondii reside in LAMP1⁺ compartments in primed macrophages. Primed mice were challenged with 4 × 10⁶ GFP-PTG i.p. Adherent PECs were stained for LAMP1. Green, GFP-PTG; red, LAMP1; blue, DAPI. In A and C, arrow heads point to lysosome-associated T. gondii with decreased GFP fluorescence. (B) Quantification of fusion events in the experiment depicted in A. Percentage of LAMP1⁺ T. gondii vacuoles (mean ± SEM) was determined by counting 20–30 T. gondii–containing vacuoles per sample (n = 3 mice per group). (C) T. gondii fuse with dextran-labeled lysosomes in primed macrophages. Green, GFP-PTG; red, dextran; blue, DAPI. (D) Quantification of fusion events in experiments depicted in C. Percentage of Texas red–positive T. gondii vacuoles was shown (the number above the bar shows the total number of T. gondii–containing vacuoles counted). (E) Bafilomycin A1 (Baf A1) treatment (100 nM) attenuated rapid parasite elimination in primed WT macrophages. Data are representative of three experiments.

Figure 3.

Ultrastructural alterations of T. gondii PVM and plasma membrane in primed WT macrophages. (A) T. gondii PVM indentation (phase I) in a primed WT macrophage. (B) Spacious PV in a primed IGTP^−/− macrophage. (C) Normal PV in a thioglycolate-elicited peritoneal macrophage. (D–K) Images of T. gondii in primed WT macrophages. (D–F) PVM vesiculation and disruption (phase II). Irregular PVM forms cuplike projections in D. ER is no longer associated with PVM undergoing vesiculation in E. PVM disintegrates into vesicles and tubules radiating away from the parasite in F. (G and H) Naked parasites in direct contact with host cytosol after PVM dissolution. Note electron-dense T. gondii plasmalemma in H. (I and J) Parasite membrane stripping (phase III). Note that the electron-dense plasmalemma has been severed, exposing the Inner membrane complex to host cytosol. Severed plasmalemma can fold onto itself, forming membrane double layers or whorls (J). (K) Detached parasite plasma membrane forming complex stacks. Note layers of detached plasmalemma held together by electron-dense bridging structures magnified in inset. T.g., T. gondii; black arrows, PVM; white arrows, PV-associated ER; black arrow heads, T. gondii plasma membrane; white arrow heads, T. gondii inner membrane complex; bars, 500 nm.

Figure 4.

Autophagic elimination of T. gondii in primed macrophages. (A) PI3K inhibitors (100 nM wortmannin, 100 μM LY294002, and 10 mM 3-MA 30 min before and after GFP-PTG infection) attenuated rapid parasite elimination in primed WT macrophages. (B) Colocalization of GFP-PTG (green) with MDC (blue) staining in primed WT macrophages at 2 h after infection. (C and D) Autophagosome formation in primed WT macrophages. Arrows point to the autophagosome outer membrane. Note the space between the outer and inner membranes and the denuded T. gondii with part of host cytoplasm (Cyt) within autophagosomes. (D) Autophagosome formation around a partially denuded parasite. Note detachment and folding of the electron-dense plasmalemma (arrow heads). (E) Fusion of flattened vesicles (arrows) giving rise to double membrane autophagosomes. (F) Fusion of lysosomes with T. gondii–containing autophagic vacuoles. Arrows point to lysosomes containing 6-nm gold particles. (G) High magnification of the fusion event shown in F. (H) EM quantification of lysosomal fusion with T. gondii. (I) Proposed phases of T. gondii elimination in primed macrophages. Black, T. gondii plasmalemma; gray, T. gondii inner membrane complex; blue, PVM; magenta, ER; green, autophagosome inner membrane; red, autophagosome outer membrane; gray vesicles, lysosomes; bars, 500 nm.

Figure 5.

Localization of IGTP in T. gondii–infected primed WT macrophages. (A) IGTP immunoreactivity (15-nm gold, highlighted by arrows) in clusters adjacent to parasite (T. gondii). (B) IGTP immunoreactivity (highlighted by arrows) associated with the periphery of an autophagosome-like profile, with the characteristic circumferential clearance around the denuded parasite (T. gondii). Arrow head pinpoints stripped parasite plasmalemma, similar to one shown in Fig. 4 C.