A Comparison of Anthrax-based Double Effector Complexes on Tumor Cell Death

Authors

• PA Bhatia
• SH Leppla

Abstract

The anthrax toxin proteins PA and LF efficiently deliver polypeptides to the cytosol of cells. The N-terminal, non-catalytic portion of LF (LFn) can be fused genetically or chemically to bacterial toxin enzymes (effectors). These effector proteins unfold and translocate in a linear manner through a protein conducting channel formed by PA. While often very potent in killing cells and in controlling tumor growth in mouse models, we asked whether greater efficacy could be achieved if several effectors were attached in tandem, a so-called “dual effector” agent. Sortase chemistry was used to attach different effectors to both LFn and full length LF. Native LF consists of LFn and a protease effector domain (LFc) that cleaves NLRP1 to cause death of macrophages in 1-2 hours. The two other effectors used were cytolethal distending toxin subunit B (CdtB) and the Pseudomonas exotoxin A catalytic domain (PEIII). Proteins containing CdtB, PEIII, or LFc strongly blocked cell growth over a period of 2-3 days. In general, the CdtB and PEIII effectors were more potent when attached to LFn than when attached to full length LF. Analyses showed that both effectors acted on substrates in the cytosol, but less efficiently when attached to full length LF. This could result from the sequential polypeptide translocation process having an inherent stochastic failure rate. For CdtB, which is believed to act in the nucleus, the larger size of LF vs LFn proteins may limit nuclear localization. These and other hypotheses will be examined in continuing studies.

Scientific Focus Area: Molecular Biology and Biochemistry

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