Comparative analysis of cytotoxic necrotizing factor (CNF) toxins: Compatibility of cargo with delivery vehicle and identification of amino acid residues that modulate pH-dependent cytosolic cargo delivery

Haywood, Elizabeth Eileen

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https://hdl.handle.net/2142/105898 Copy

Description

Title

Comparative analysis of cytotoxic necrotizing factor (CNF) toxins: Compatibility of cargo with delivery vehicle and identification of amino acid residues that modulate pH-dependent cytosolic cargo delivery

Author(s)

Haywood, Elizabeth Eileen

Issue Date

2019-07-03

Director of Research (if dissertation) or Advisor (if thesis)

Wilson, Brenda A

Doctoral Committee Chair(s)

Wilson, Brenda A

Committee Member(s)

• Blanke, Steven R.
• Kehl-Fie, Thomas E.
• Imlay, James A.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• AB toxin
• Cargo delivery vehicle
• CNF
• Cytotoxic necrotizing factor
• Intracellular delivery
• Bacterial toxin-inspired drug delivery
• Biologics

Abstract

AB toxins are modular proteins that deliver their catalytic cargo into the cytosol of host cells. These toxins are increasingly being exploited for targeted delivery in biomedical and research applications due to their potency and cell specificity. This thesis explores the suitability of the cytotoxic necrotizing factor (CNF) family as a model system for the development of Bacterial Toxin-Inspired Drug Delivery (BTIDD) platforms. The CNF family is notable among AB toxins in that there are nine full-length homologs and many CNF-like catalytic domains associated with various other putative delivery systems. The data presented herein demonstrates how cargo and delivery modules of the CNF family members, CNF1, CNF2, CNF3, and CNFy, can be assembled to maintain efficient biological activity, and refines the joining sites for assembly of chimeric toxins to enhance their delivery efficiency. Through cell-based luciferase reporter assays, we show that the CNFy cargo domain is the most universally compatible and that the CNF3 delivery vehicle is the most flexible and efficient at delivering non-native cargo. Further, we show that the CNF3 delivery domain delivers the CNF2 and CNFy cargos more efficiently than their native delivery vehicles. We also investigated whether the previously reported differential sensitivity of the CNF toxins to endosomal acidification impacts their cargo delivery efficiency. We found that replacing particular acidic amino acid residues from the putative insertion-trigger motif of the CNFy translocation domain with those in CNF3 promotes endosomal escape at a higher pH, leading to more efficient cytosolic delivery. These findings provide insight into the amino acid determinants of bacterial toxins that can be exploited to optimize cytosolic delivery of biologic cargos using BTIDD platforms.

Graduation Semester

2019-08

Type of Resource

text

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Copyright and License Information

Copyright 2019 Elizabeth Eileen Haywood

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