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Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease
Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the dev...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3994088/ https://www.ncbi.nlm.nih.gov/pubmed/24752186 http://dx.doi.org/10.1371/journal.pone.0095574 |
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author | Kiedrowski, Megan R. Crosby, Heidi A. Hernandez, Frank J. Malone, Cheryl L. McNamara, James O. Horswill, Alexander R. |
author_facet | Kiedrowski, Megan R. Crosby, Heidi A. Hernandez, Frank J. Malone, Cheryl L. McNamara, James O. Horswill, Alexander R. |
author_sort | Kiedrowski, Megan R. |
collection | PubMed |
description | Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme. |
format | Online Article Text |
id | pubmed-3994088 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39940882014-04-25 Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease Kiedrowski, Megan R. Crosby, Heidi A. Hernandez, Frank J. Malone, Cheryl L. McNamara, James O. Horswill, Alexander R. PLoS One Research Article Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme. Public Library of Science 2014-04-21 /pmc/articles/PMC3994088/ /pubmed/24752186 http://dx.doi.org/10.1371/journal.pone.0095574 Text en © 2014 Kiedrowski et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Kiedrowski, Megan R. Crosby, Heidi A. Hernandez, Frank J. Malone, Cheryl L. McNamara, James O. Horswill, Alexander R. Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease |
title |
Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease |
title_full |
Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease |
title_fullStr |
Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease |
title_full_unstemmed |
Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease |
title_short |
Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease |
title_sort | staphylococcus aureus nuc2 is a functional, surface-attached extracellular nuclease |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3994088/ https://www.ncbi.nlm.nih.gov/pubmed/24752186 http://dx.doi.org/10.1371/journal.pone.0095574 |
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