Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins

Elizabeth E Haywood,Nicholas B Handy,James W Lopez,Mengfei Ho,Brenda A Wilson

doi:10.1016/j.jbc.2021.101347

Elizabeth E Haywood, Nicholas B Handy + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101347

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Abstract

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.

Highlights

CNF1 into cells, CNFy does not bind LPR, Modular bacterial toxins deliver their catalytic cargo into the cytosol of specific target cells
EGA blocks trafficking from the the translocation region of CNF1 to the so-called early endosome to the late endosome (30-32), Journal Pre-proof “dagger” membrane-insertion motif
Our results indicate that CNF3 is the most resistant to NH4Cl treatment, while CNFy is the most sensitive, and CNF1 and CNF2 are intermediate, suggesting that CNF3 escapes the endosome at a higher pH than the others, and CNFy requires more acidic late endosomes for escape

Summary

INTRODUCTION

CNF1 into cells, CNFy does not bind LPR, Modular bacterial toxins deliver their catalytic cargo into the cytosol of specific target cells. TH8-TH9) found in the T domain of diphtheria toxin (DT) (27,28), a model for the pH-dependent insertion step of the cargo-delivery process was previously proposed involving a putative helixloop-helix (HLH) region (residues [350-412] in CNF1) (24) This putative HLH of CNF1 contains four highly conserved acidic residues (D373, D379, E382, E383) in the postulated loop region that were proposed to become protonated in the acidic environment of the late endosome, thereby allowing insertion as a “dagger” into the membrane. Site-specific as NH4Cl that acts as a weak base to raise the mutational analysis identified two acidic residues endosomal pH and bafilomycin A1 that blocks within this region responsible for mediating the acidification by inhibition of the vacuolar differential sensitivities to NH4Cl. CNF3 and ATPase proton pump. That these two toxins have the greatest sequence homology and are identical regarding charged

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES

Loop H2