Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins

Abstract Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a ke...

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Autores principales:	Anna Lepesheva, Adriana Osickova, Jana Holubova, David Jurnecka, Sarka Knoblochova, Carlos Espinosa-Vinals, Ladislav Bumba, Karolina Skopova, Radovan Fiser, Radim Osicka, Peter Sebo, Jiri Masin
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6f873568b2e4490a9da5948d8fb80b36
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spelling	oai:doaj.org-article:6f873568b2e4490a9da5948d8fb80b362021-12-02T18:37:10ZDifferent roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins10.1038/s41598-021-99112-32045-2322https://doaj.org/article/6f873568b2e4490a9da5948d8fb80b362021-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-99112-3https://doaj.org/toc/2045-2322Abstract Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.Anna LepeshevaAdriana OsickovaJana HolubovaDavid JurneckaSarka KnoblochovaCarlos Espinosa-VinalsLadislav BumbaKarolina SkopovaRadovan FiserRadim OsickaPeter SeboJiri MasinNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anna Lepesheva Adriana Osickova Jana Holubova David Jurnecka Sarka Knoblochova Carlos Espinosa-Vinals Ladislav Bumba Karolina Skopova Radovan Fiser Radim Osicka Peter Sebo Jiri Masin Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins
description	Abstract Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.
format	article
author	Anna Lepesheva Adriana Osickova Jana Holubova David Jurnecka Sarka Knoblochova Carlos Espinosa-Vinals Ladislav Bumba Karolina Skopova Radovan Fiser Radim Osicka Peter Sebo Jiri Masin
author_facet	Anna Lepesheva Adriana Osickova Jana Holubova David Jurnecka Sarka Knoblochova Carlos Espinosa-Vinals Ladislav Bumba Karolina Skopova Radovan Fiser Radim Osicka Peter Sebo Jiri Masin
author_sort	Anna Lepesheva
title	Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins
title_short	Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins
title_full	Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins
title_fullStr	Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins
title_full_unstemmed	Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins
title_sort	different roles of conserved tyrosine residues of the acylated domains in folding and activity of rtx toxins
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/6f873568b2e4490a9da5948d8fb80b36
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Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins

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