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Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate int...

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Autores principales: Bland, David M., Eisele, Nicholas A., Keleher, Lauren L., Anderson, Paul E., Anderson, Deborah M.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047566/
https://www.ncbi.nlm.nih.gov/pubmed/21399698
http://dx.doi.org/10.1371/journal.pone.0017352
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author Bland, David M.
Eisele, Nicholas A.
Keleher, Lauren L.
Anderson, Paul E.
Anderson, Deborah M.
author_facet Bland, David M.
Eisele, Nicholas A.
Keleher, Lauren L.
Anderson, Paul E.
Anderson, Deborah M.
author_sort Bland, David M.
collection PubMed
description Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence.
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spelling pubmed-30475662011-03-11 Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis Bland, David M. Eisele, Nicholas A. Keleher, Lauren L. Anderson, Paul E. Anderson, Deborah M. PLoS One Research Article Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence. Public Library of Science 2011-03-02 /pmc/articles/PMC3047566/ /pubmed/21399698 http://dx.doi.org/10.1371/journal.pone.0017352 Text en Bland 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
Bland, David M.
Eisele, Nicholas A.
Keleher, Lauren L.
Anderson, Paul E.
Anderson, Deborah M.
Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
title Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
title_full Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
title_fullStr Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
title_full_unstemmed Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
title_short Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis
title_sort novel genetic tools for diaminopimelic acid selection in virulence studies of yersinia pestis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047566/
https://www.ncbi.nlm.nih.gov/pubmed/21399698
http://dx.doi.org/10.1371/journal.pone.0017352
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