Figure 1.
An active site affinity tag identifies processed active proteases of wild type and cruzain deficient T. cruzi.
Iodinated DCGO4 (0.1, 1, 10, and-50 µM) was reacted with epimastigote extracts (105 epimastigotes/lane). Processed, active cruzain (47.5 kD) was identified as the major protease in wild type T. cruzi. Higher molecular species identified as unprocessed cruzain by MALDI but no active protease were observed in cruzain-deficient parasites at 50 µM DCG04.
Figure 2.
IEM confirms lack of surface-associated cruzain.
A cruzain-specific gold-labeled secondary antibody identified cruzain on the cell surface of intracellular wild type and cruzain-deficient T. cruzi amastigotes. A 3-fold (p<0.1) reduction in gold-labeled Ab detection of cruzain was present on the cell surface and flagellar pocket of cruzain-deficient CA-I/KR (B) versus wild type amastigotes (A) (45,000X). Results are representative of three independent experiments. N, nucleus; K, kinetoplast; FP, flagellar pocket; C, cytoplasm of BESM cell.
Figure 3.
Cruzain-deficient (KR) but not WT parasites activate NF-κB.
Uninfected controls and mouse macrophages (MΦ) infected with WT or KR T. cruzi were infected for ≤ 1 min (lanes 1, 2), 30 minutes (lanes 3–7) and 60 minutes (lanes 8–11). Cell extracts were western blotted with: A, anti-NF-κB P65 Ab; B, anti ∼P-iκB; C, anti-actin Ab. MΦ were infected as follows: lane #1, WT; lane #2, KR; lane #3, uninfected MΦ; lane #4, WT; lane #5, KR; lane #6, KR with 10 µM K11777; lane #7, KR cultured without K777 for 2 months; lane #8, WT; lane #9, KR; lane #10, KR with 10 µM K11777; lane #11, KR cultured without K11777 for 2 months. Results are representative of six independent experiments using two different methods. Controls (D) were MΦ treated or not with K11777 and/or LPS.
Figure 4.
NF-κB P65 localizes to the cell surface of wild type parasites.
Localization of NF-κB P65 (green fluorescence) was investigated in mouse macrophages. Parasite and host cell DNA was labeled with PI (red). Non-infected macrophages showed an evenly scattered distribution of cytoplasmic NF-κB P65 (E). Intracellular WT- T. cruzi appeared coated with macrophage NF-κB P65 (A, B). In contrast, abundant cytoplasmic (green fluorescence) and nuclear (yellow fluorescence) NF-κB P65 occurred in macrophages infected with cruzain deficient T. cruzi (C–D). Macrophages were infected for 30 min (A, C), and 60 min (B, D). Results are representative of four independent experiments.
Figure 5.
Colocalization of cruzain and NF-κB P65.
A–C. Representative examples of colocalization of labeled cruzain (A) (red fluorescence) and NF-κB P65 (B) (green fluorescence) in the flagellar pocket region and/or cell surface of interiorized transforming parasites (C, merge). D. Lack of colocalization of accumulated cruzain (red fluorescence) and NF-κB P65 (green) in a macrophage infected as above with WT T. cruzi and treated with 10 µM K11777 that induces accumulation of unprocessed zymogen in the Golgi compartment (arrow) (22).
Figure 6.
Proteolytic cleavage of r-human NF-κB P65 by wild type- and r-cruzain but not by proteases expressed by cruzain-deficient T. cruzi.
Recombinant human NF-κB P65 was treated or not with cruzain or parasite extracts. Lane 1, untreated control; lane 2, r-cruzain (1∶1 molar ratio); lanes 3–4: dilutions 1∶10 and 1∶100 of r-cruzain, respectively; lane 5, r-cruzain (1∶1 molar ratio) and 10 µM K11777; lane 6, WT cruzain; lane 7, proteases in cruzain-deficient T. cruzi. Results are representative of two independent experiments.
Figure 7.
Macrophage activation prevents T. cruzi intracellular development.
Mouse macrophages were infected with wild type T. cruzi and LPS was added as indicated. Mean parasites/cell were determined at 1 h and 48 h post-infection. Parasite death occurred when LPS was added prior to and concomitantly with T. cruzi trypomastigotes. Results are representative of two independent experiments (n = 3 per treatment).
Figure 8.
No significant IL12 expression was detected in macrophages infected with wild type T. cruzi while cells treated with 10 µM K11777 had significantly higher cytoplasmic IL12 levels. Increased IL12 expression also occurred in macrophages infected with cruzain-deficient T. cruzi. Results are representative of three independent experiments. A. Wild type-infected control. B. Macrophage infected wild type T. cruzi and treated with 10 µM K11777. C. Macrophage infected with cruzain-deficient T. cruzi.