From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors

Zuverink, Madison; Barbieri, Joseph T.

doi:10.1093/femspd/ftv097

Cited by 6 publications

(11 citation statements)

References 63 publications

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“…␤lac-TT was developed as a reporter for a cell-based model of LC translocation by tetanus toxin detected as the cytosolic cleavage of a FRET-based substrate, CCF2 (12). Overall, this assay has proven to be a useful discoverybased system (13). In initial experiments, two conserved regions of the HCN, a hydrophobic region within ␣12 and a charged loop between ␣15 and ␣16 (cis-loop) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Tetanus Toxin cis -Loop Contributes to Light-Chain Translocation

Zuverink

Bluma

Barbieri

2020

mSphere

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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.

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Section: Resultsmentioning

confidence: 99%

Tetanus Toxin cis -Loop Contributes to Light-Chain Translocation

Zuverink

Bluma

Barbieri

2020

mSphere

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“…The biologic relevance of the interactions detected in vitro with the AIP56 transport domain was investigated using a beta-lactamase (Bla)-AIP56 258–497 chimera, in which the catalytic domain of AIP56 was replaced by Bla 19–286 . This chimera was incubated with mBMDM, and the arrival of the Bla moiety at the cytosol was evaluated by a FRET-based assay using the CCF4-AM substrate 33 . This fluorescent substrate consists of a cephalosporin backbone that connects coumarin and fluorescein molecules.…”

Section: Resultsmentioning

confidence: 99%

Involvement of Hsp90 and cyclophilins in intoxication by AIP56, a metalloprotease toxin from Photobacterium damselae subsp. piscicida

Rodrigues

Pereira

Lisboa

et al. 2019

Sci Rep

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AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida ( Phdp ), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-κB p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile . Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.

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“…Colors represent the fluorescent signal recorded before (left) and after (right) the effector translocation has started. Discussion on the pros and cons of some of these assays can be found in Ehsani et al ( 2009 ) and Zuverink and Barbieri ( 2015 ).…”

Section: Identification Of Secreted Proteinsmentioning

confidence: 99%

“…Effector proteins are fused to beta-lactamase, while host cells are pre-loaded with the membrane-permeable substrate coumarin cephalosporin fluorescein (CCF2 or CCF4). The injection of the effector/beta-lactamase fusion protein into the host cytosol is detected by the loss of FRET upon cleavage of the fluorogenic substrate, inducing a switch in the fluorescence from green to blue (Charpentier and Oswald, 2004 ; Zuverink and Barbieri, 2015 ; Figure 5 ). This system has for instance been used with the beta-lactamase reporter encoded chromosomally in fusion to twenty different enteropathogenic E. coli (EPEC) effectors (Mills et al, 2013 ).…”

Section: Resolution Of Secretion In Time and Spacementioning

confidence: 99%

Tracking Proteins Secreted by Bacteria: What's in the Toolbox?

Maffei

Francetić

Subtil

2017

Front. Cell. Infect. Microbiol.

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Bacteria have acquired multiple systems to expose proteins on their surface, release them in the extracellular environment or even inject them into a neighboring cell. Protein secretion has a high adaptive value and secreted proteins are implicated in many functions, which are often essential for bacterial fitness. Several secreted proteins or secretion machineries have been extensively studied as potential drug targets. It is therefore important to identify the secretion substrates, to understand how they are specifically recognized by the secretion machineries, and how transport through these machineries occurs. The purpose of this review is to provide an overview of the biochemical, genetic and imaging tools that have been developed to evaluate protein secretion in a qualitative or quantitative manner. After a brief overview of the different tools available, we will illustrate their advantages and limitations through a discussion of some of the current open questions related to protein secretion. We will start with the question of the identification of secreted proteins, which for many bacteria remains a critical initial step toward a better understanding of their interactions with the environment. We will then illustrate our toolbox by reporting how these tools have been applied to better understand how substrates are recognized by their cognate machinery, and how secretion proceeds. Finally, we will highlight recent approaches that aim at investigating secretion in real time, and in complex environments such as a tissue or an organism.

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From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors

Cited by 6 publications

References 63 publications

Tetanus Toxin cis -Loop Contributes to Light-Chain Translocation

Tetanus Toxin cis -Loop Contributes to Light-Chain Translocation

Involvement of Hsp90 and cyclophilins in intoxication by AIP56, a metalloprotease toxin from Photobacterium damselae subsp. piscicida

Tracking Proteins Secreted by Bacteria: What's in the Toolbox?

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