Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction

Abigail L Male,Francesco Cuda,Gong Zhang,Siqi Zheng,Petra C F Oyston,Ali Tavassoli,Peng R Chen,E Diane Williamson,Fedor Forafonov

doi:10.1038/s41598-017-03253-3

Abstract

The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.

Highlights

Anthrax is caused by Bacillus anthracis (B. anthracis), a spore-forming encapsulated Gram positive bacterium[1, 2]
We employed a genetically encoded, high-throughput screening platform that combines a bacterial reverse two-hybrid system (RTHS)[21, 22] to screen a library of 3.2 million cyclic peptides generated by split-intein circular ligation of peptides and proteins (SICLOPPS)[23,24,25]
protective antigen (PA) is composed of four distinct domains, termed domain I– IV (Supplemental Figure 1A)[26, 27]; three isopropyl β-D-1-thiogalactopyranoside (IPTG) induced plasmids each encoding full length PA, domain II to domain IV of PA259–735, or domain III and IV of PA488–735 as an N-terminal fusion with the 434 repressor, and the extracellular portion of CMG238–218 as an N-terminal fusion with a chimeric P22 repressor were constructed

Summary

Introduction

Anthrax is caused by Bacillus anthracis (B. anthracis), a spore-forming encapsulated Gram positive bacterium[1, 2]. PA is an 83 kDa protein that binds to one of two cell surface receptors, undergoes furin protease-mediated cleavage to yield a 63 kDa fragment. This cleavage is essential for toxin action and PA harbouring mutations in the furin cleavage site is completely non-toxic and devoid of pathogenic effects in vivo[6]. A series of elegant experiments with mice lacking TEM8, CMG2 or both, have demonstrated that the main receptor for anthrax toxicity is CMG211 This may be due to the higher affinity of PA for CMG2 (170 pM) versus TEM8 (1.1 μM)[12], or the fact that CMG2 is preferentially expressed in cells important for infection and/or toxin-induced death[13]. We describe the identification and in vitro validation of a linear pentapeptide that binds CMG2 and inhibits the protein-protein interaction (PPI) with PA

Methods

Results

Conclusion