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Brucella dissociation is essential for macrophage egress and bacterial dissemination

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed...

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Autores principales: Pei, Jianwu, Kahl-McDonagh, Melissa, Ficht, Thomas A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942807/
https://www.ncbi.nlm.nih.gov/pubmed/24634889
http://dx.doi.org/10.3389/fcimb.2014.00023
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author Pei, Jianwu
Kahl-McDonagh, Melissa
Ficht, Thomas A.
author_facet Pei, Jianwu
Kahl-McDonagh, Melissa
Ficht, Thomas A.
author_sort Pei, Jianwu
collection PubMed
description It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4–5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.
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spelling pubmed-39428072014-03-14 Brucella dissociation is essential for macrophage egress and bacterial dissemination Pei, Jianwu Kahl-McDonagh, Melissa Ficht, Thomas A. Front Cell Infect Microbiol Microbiology It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4–5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination. Frontiers Media S.A. 2014-03-05 /pmc/articles/PMC3942807/ /pubmed/24634889 http://dx.doi.org/10.3389/fcimb.2014.00023 Text en Copyright © 2014 Pei, Kahl-McDonagh and Ficht. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Pei, Jianwu
Kahl-McDonagh, Melissa
Ficht, Thomas A.
Brucella dissociation is essential for macrophage egress and bacterial dissemination
title Brucella dissociation is essential for macrophage egress and bacterial dissemination
title_full Brucella dissociation is essential for macrophage egress and bacterial dissemination
title_fullStr Brucella dissociation is essential for macrophage egress and bacterial dissemination
title_full_unstemmed Brucella dissociation is essential for macrophage egress and bacterial dissemination
title_short Brucella dissociation is essential for macrophage egress and bacterial dissemination
title_sort brucella dissociation is essential for macrophage egress and bacterial dissemination
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942807/
https://www.ncbi.nlm.nih.gov/pubmed/24634889
http://dx.doi.org/10.3389/fcimb.2014.00023
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