Engineering Photorhabdus luminescens toxin complex (PTC) into a recombinant injection nanomachine

Abstract

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe-like nanomachine for delivery of protein toxins from different species. In addition, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the probable size limitation, the charge of the cargo also influences the efficiency of packing and transport into mammalian cells. Our data show that the PTC constitutes a powerful system to inject recombinant proteins, peptides, and potentially, other molecules into mammalian cells. In addition, in contrast to other protein transporters based on pore formation, the closed, compact structure of the PTC may protect cargo from degradation.

Figure 1.. Activity of BC3-C3bot fusion toxin.

(A) Design of the fusion toxin. The TcC3 C-terminal hvr was replaced with a selected cargo C terminus to the auto proteolytic cleavage site (grey box). This cargo would then be packaged into the BC3 cocoon (grey oval within the blue) (adapted from Meusch et al, 2014). (B) The purified BC3-C3bot fusion protein was separated on an SDS–PAGE gel (Coomassie blue). For the Western blot, a C3bot-specific antibody was used (Aktories et al, 1989). (C) Autoradiogram of in vitro ADP-ribosylation of RhoA by 3 nM of BC3-C3bot and 1.25 nM C3bot (WT). (D) Intoxication of HeLa cells with 10 nM of TcA + BC3-C3bot each for 6 h at 37°C. (E) Quantification of intoxication of HeLa cells treated as in (D), plus 100 nM of C3bot (WT). (F) TEER assay of CaCo-2 cells treated with 1.94 nM of TcA and 5.1 nM of BC3-C3bot for 8 h. Unpaired, two-tailed t test (P < 0.001) (±SEM). Scale bar: 100 μm. N = 3.

Figure S1.. Effect of temperature on the BC3 cocoon.

Samples were incubated at the indicated temperatures for 1, 5, and 10 min, cooled, and the assay conducted at 21°C. (A, B) BC3 (WT) (B) TccC3hvr. Unpaired, two-tailed t test (P < 0.05) (±SEM). N = 3.

Figure 2.. Activity of BC3-YopT fusion toxins.

(A) In vitro membrane release activity of RhoA from purified HeLa cell membranes by BC3-YopT chimeras. (B) In vivo activity of BC3-YopT. HeLa cells were intoxicated either overnight (top two panels) or for 4 h with TcA + BC3-YopTs (20 nM) (bottom two panels), then 1 h with CNF1 (4 nM) before being fixed and actin stained. (C) Biochemical analysis of in vivo membrane release of RhoA by BC3-YopT^full in HeLa cells intoxicated as in B above. Scale bar: 100 μm. N = 3. P, Pellet (membrane fraction); S, post-membrane supernatant.

Figure 3.. In vivo activity of BC3-YopT fusion toxins.

Confluent monolayers of CaCo-2 cells were intoxicated with increasing concentrations of TcA + BC3-YopT fusion toxins and TEER was measured. The graphs show TEER as a percentage of the starting value. (A) BC3-YopT^Δ1−74. (B) BC3-YopT^Δ1−74-lys3. (C) BC3-YopT^Δ1−74-lys6. (D) BC3-YopT^Δ1−30. (E) BC3-YopT^full. (F) A comparison of all toxins (30 nM each). Recombinant PTC3 was used as a positive control. N = 3.

Figure S2.. Calculation of EC50 values.

EC50 calculation of BC3-YopT fusion toxins after intoxication of CaCo-2 cells with increasing concentrations of TcA + BC3-YopTs for 20 h. BC3-YopT^Δ1−74-lys3 (yellow) = 17.43, BC3-YopT^Δ1−74-lys6 (blue) = 19.7, BC3-YopT^Δ1−30 (green) = 8.27, and BC3-YopT^full (red) = 14.52. N = 3.

Figure 4.. Luciferase activity of BC3-MLuc7 fusion toxin.

(A) In vivo activity of MLuc7 after treatment of HeLa cells with 10 nM of TcA + BC3-MLuc7 for 1 h at 37°C. (B) pH-dependent delivery of MLuc7 across the cell membrane of HeLa cells. (C) In vitro activity of BC3-MLuc7 in passive lysis buffer (Buffer) or HeLa cell lysate. (D) pH-dependent delivery of MLuc7 across the cell membrane at pH 7.5. (E) pH-dependent delivery at pH 5. Unpaired, two-tailed t test (P < 0.01) (±SEM). N = 3. RLU, relative luminescence units (luminescence per μg of total protein).

Figure S3.. Effect of endocytosis inhibition on the efficiency of cargo delivery by the PTC.

(A) Intoxication of HeLa cells by the recombinant wild-type PTC3 (1.4 nM TcA + 3.5 nM BC3) in the presence/absence of nocodazole. (B) Delivery of MLuc7 across the plasma membrane of HeLa cells by PTC3-MLuc7 (3 nM of TcA + BC3-MLuc7) in the presence/absence of nocodazole (20 μM) at pH 5. Unpaired, two-tailed t test (P < 0.01) (±SEM). N = 3. RLU, relative luminescence units (luminescence per μg of total protein).

Figure S4.. BC3-MLuc7 activity and quantification of the efficiency of PTC3-MLuc7.

(A) Linear regression calculation of BC3-MLuc7 luciferase activity in HeLa cell lysate. (B) Normality test of data used in (A). (C) In vitro activity of BC3-MLuc7 in different buffers and enzyme treatments. ±SEM. N = 3.

Figure 5.. Bioluminescence activity of BC3-MLuc7 fusion toxin.

Membrane delivery of toxin (10 nM) into HeLa cells was performed for 1 h at 4°C. Then, the cells were transferred to a live cell imaging microscope, where 2 μg/ml of coelenterazine H was added before immediate visualization for 10 min. The controls include coelenterazine only, TcA + coelenterazine, and BC3-MLuc7 + coelenterazine. Figure represents one of two independent experiments. Scale bar: 15 μm. BL, bioluminescence; DIC, differential interference contrast.