A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants
Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R)...
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doaj-670ad39db8314a73b20d62223958adc42020-11-25T00:34:21ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882018-03-01810.3389/fcimb.2018.00069342347A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid MutantsAlbertus Viljoen0Ana Victoria Gutiérrez1Ana Victoria Gutiérrez2Christian Dupont3Eric Ghigo4Laurent Kremer5Laurent Kremer6Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Montpellier, FranceCentre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Montpellier, FranceUnité de Recherche Microbes, Evolution, Phylogeny and Infection (MEPHI), Institut Hospitalier Universitaire Méditerranée-Infection, Marseille, FranceCentre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Montpellier, FranceCentre National de la Recherche Scientifique, Marseille, FranceCentre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Montpellier, FranceIRIM, 34293, Institut National de la Santé et de la Recherche Médicale, Montpellier, FranceLittle is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective.http://journal.frontiersin.org/article/10.3389/fcimb.2018.00069/fullgene disruptionMycobacterium abscessuszebrafishvirulenceglycopeptidolipid |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Albertus Viljoen Ana Victoria Gutiérrez Ana Victoria Gutiérrez Christian Dupont Eric Ghigo Laurent Kremer Laurent Kremer |
spellingShingle |
Albertus Viljoen Ana Victoria Gutiérrez Ana Victoria Gutiérrez Christian Dupont Eric Ghigo Laurent Kremer Laurent Kremer A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants Frontiers in Cellular and Infection Microbiology gene disruption Mycobacterium abscessus zebrafish virulence glycopeptidolipid |
author_facet |
Albertus Viljoen Ana Victoria Gutiérrez Ana Victoria Gutiérrez Christian Dupont Eric Ghigo Laurent Kremer Laurent Kremer |
author_sort |
Albertus Viljoen |
title |
A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants |
title_short |
A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants |
title_full |
A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants |
title_fullStr |
A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants |
title_full_unstemmed |
A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants |
title_sort |
simple and rapid gene disruption strategy in mycobacterium abscessus: on the design and application of glycopeptidolipid mutants |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cellular and Infection Microbiology |
issn |
2235-2988 |
publishDate |
2018-03-01 |
description |
Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus, increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus (M. abscessus) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus, testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus, we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a, necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of M. abscessus, potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective. |
topic |
gene disruption Mycobacterium abscessus zebrafish virulence glycopeptidolipid |
url |
http://journal.frontiersin.org/article/10.3389/fcimb.2018.00069/full |
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