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Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three...

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Autores principales: Donnelly, M. Lauren, Forster, Emily R., Rohlfing, Amy E., Shen, Aimee
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200643/
https://www.ncbi.nlm.nih.gov/pubmed/32242623
http://dx.doi.org/10.1042/BCJ20190875
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author Donnelly, M. Lauren
Forster, Emily R.
Rohlfing, Amy E.
Shen, Aimee
author_facet Donnelly, M. Lauren
Forster, Emily R.
Rohlfing, Amy E.
Shen, Aimee
author_sort Donnelly, M. Lauren
collection PubMed
description Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently solved crystal structure of CspC revealed that its pseudoactive site residues align closely with the catalytic triad of CspB, suggesting that it might be possible to ‘resurrect' the ancestral protease activity of the CspC and CspA pseudoproteases. Here, we demonstrate that restoring the catalytic triad to these pseudoproteases fails to resurrect their protease activity. We further show that the pseudoactive site substitutions differentially affect the stability and function of the CspC and CspA pseudoproteases: the substitutions destabilized CspC and impaired spore germination without affecting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results indicate that bioinformatic predictions of enzyme activity may underestimate pseudoenzymes in rare cases.
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spelling pubmed-72006432020-05-13 Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination Donnelly, M. Lauren Forster, Emily R. Rohlfing, Amy E. Shen, Aimee Biochem J Microbiology Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently solved crystal structure of CspC revealed that its pseudoactive site residues align closely with the catalytic triad of CspB, suggesting that it might be possible to ‘resurrect' the ancestral protease activity of the CspC and CspA pseudoproteases. Here, we demonstrate that restoring the catalytic triad to these pseudoproteases fails to resurrect their protease activity. We further show that the pseudoactive site substitutions differentially affect the stability and function of the CspC and CspA pseudoproteases: the substitutions destabilized CspC and impaired spore germination without affecting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results indicate that bioinformatic predictions of enzyme activity may underestimate pseudoenzymes in rare cases. Portland Press Ltd. 2020-04-30 2020-04-27 /pmc/articles/PMC7200643/ /pubmed/32242623 http://dx.doi.org/10.1042/BCJ20190875 Text en © 2020 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Microbiology
Donnelly, M. Lauren
Forster, Emily R.
Rohlfing, Amy E.
Shen, Aimee
Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination
title Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination
title_full Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination
title_fullStr Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination
title_full_unstemmed Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination
title_short Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination
title_sort differential effects of ‘resurrecting' csp pseudoproteases during clostridioides difficile spore germination
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200643/
https://www.ncbi.nlm.nih.gov/pubmed/32242623
http://dx.doi.org/10.1042/BCJ20190875
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