Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation

ABSTRACT The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chai...

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Autores principales:	Madison Zuverink, Matthew Bluma, Joseph T. Barbieri
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Clostridium cell biology exotoxins protein translocation tetanus toxins Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/609bbce3fe9c4dd183063eb35ca4ce62
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spelling	oai:doaj.org-article:609bbce3fe9c4dd183063eb35ca4ce622021-11-15T15:30:15ZTetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation10.1128/mSphere.00244-202379-5042https://doaj.org/article/609bbce3fe9c4dd183063eb35ca4ce622020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00244-20https://doaj.org/toc/2379-5042ABSTRACT The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation. IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.Madison ZuverinkMatthew BlumaJoseph T. BarbieriAmerican Society for MicrobiologyarticleClostridiumcell biologyexotoxinsprotein translocationtetanustoxinsMicrobiologyQR1-502ENmSphere, Vol 5, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridium cell biology exotoxins protein translocation tetanus toxins Microbiology QR1-502
spellingShingle	Clostridium cell biology exotoxins protein translocation tetanus toxins Microbiology QR1-502 Madison Zuverink Matthew Bluma Joseph T. Barbieri Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation
description	ABSTRACT The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation. IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.
format	article
author	Madison Zuverink Matthew Bluma Joseph T. Barbieri
author_facet	Madison Zuverink Matthew Bluma Joseph T. Barbieri
author_sort	Madison Zuverink
title	Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation
title_short	Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation
title_full	Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation
title_fullStr	Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation
title_full_unstemmed	Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation
title_sort	tetanus toxin <italic toggle="yes">cis</italic>-loop contributes to light-chain translocation
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/609bbce3fe9c4dd183063eb35ca4ce62
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Tetanus Toxin <italic toggle="yes">cis</italic>-Loop Contributes to Light-Chain Translocation

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