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Identification of commensal Escherichia coli genes involved in biofilm resistance to pathogen colonization.

Da Re S, Valle J, Charbonnel N, Beloin C, Latour-Lambert P, Faure P, Turlin E, Le Bouguénec C, Renauld-Mongénie G, Forestier C, Ghigo JM - PLoS ONE (2013)

Bottom Line: We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm.We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut.This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Génétique des Biofilms, Département de Microbiologie, Paris, France.

ABSTRACT
Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

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Constitutive expression of potential colonization resistance genes.Estimate of E. coli 55989a (P) colonization in mixed biofilms with wild-type E. coli MG1655 F′ (C), corresponding deletion mutants (MG1655Δgene: C*) or overexpressed (MG1655PcLgene: C+) derivative strains. Results are represented as ratio of colonization l of mutant mixed biofilms to wild-type mixed C+P biofilms. Black bar represents wild-type colonization in C+P mixed biofilms arbitrarily set to one. White bars represent colonization of pathogen in mixed CΔgene + P biofilms. Stripped bars show pathogen colonization in mixed CPcLgene + P biofilms with commensal overexpressing potential colonization resistance genes. Genes deleted or overexpressed are indicated under the bars. Results are averages of at least 12 replicates ± standard deviation of the mean. The extent of colonization in CΔgene + P mixed biofilm was significantly different from that of wild-type C+P biofilm p<0.05; asterisks indicate significant difference between extent of colonization in over-expressed and deletion mutants, p<0.05.
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pone-0061628-g002: Constitutive expression of potential colonization resistance genes.Estimate of E. coli 55989a (P) colonization in mixed biofilms with wild-type E. coli MG1655 F′ (C), corresponding deletion mutants (MG1655Δgene: C*) or overexpressed (MG1655PcLgene: C+) derivative strains. Results are represented as ratio of colonization l of mutant mixed biofilms to wild-type mixed C+P biofilms. Black bar represents wild-type colonization in C+P mixed biofilms arbitrarily set to one. White bars represent colonization of pathogen in mixed CΔgene + P biofilms. Stripped bars show pathogen colonization in mixed CPcLgene + P biofilms with commensal overexpressing potential colonization resistance genes. Genes deleted or overexpressed are indicated under the bars. Results are averages of at least 12 replicates ± standard deviation of the mean. The extent of colonization in CΔgene + P mixed biofilm was significantly different from that of wild-type C+P biofilm p<0.05; asterisks indicate significant difference between extent of colonization in over-expressed and deletion mutants, p<0.05.

Mentions: We then used a previously described plasmid-free expression strategy and placed a constitutive promoter in front of the yiaF, stfE, yliE and ypjC genes directly on the MG1655 F′ chromosome [34], [40]. This insertion had no effect on growth or biofilm formation for yiaF, stfE, yliE and ypjC mutant strains (data not shown). However, constitutive expression of rcsA (MG1655PcLrbs-rcsA F′) led to mucoidy and the inability to form biofilm (data not shown), and this mutant was not further analyzed. The colonization phenotype of the 4 remaining mutants was evaluated and compared to that of the corresponding deletion mutants. While constitutive expression of ypjC and stfE did not lead to significant 55989a colonization changes, yiaF and yliE constitutive expression in MG1655PcLrbs-yiaF F′ and MG1655PcLrbs-yliE F′, respectively, significantly reduced the ability of E. coli pathogen 55989a to colonize the resulting C+P mixed biofilm compared to colonization of the corresponding MG1655 F′ deletion mutants (Fig. 2).


Identification of commensal Escherichia coli genes involved in biofilm resistance to pathogen colonization.

Da Re S, Valle J, Charbonnel N, Beloin C, Latour-Lambert P, Faure P, Turlin E, Le Bouguénec C, Renauld-Mongénie G, Forestier C, Ghigo JM - PLoS ONE (2013)

Constitutive expression of potential colonization resistance genes.Estimate of E. coli 55989a (P) colonization in mixed biofilms with wild-type E. coli MG1655 F′ (C), corresponding deletion mutants (MG1655Δgene: C*) or overexpressed (MG1655PcLgene: C+) derivative strains. Results are represented as ratio of colonization l of mutant mixed biofilms to wild-type mixed C+P biofilms. Black bar represents wild-type colonization in C+P mixed biofilms arbitrarily set to one. White bars represent colonization of pathogen in mixed CΔgene + P biofilms. Stripped bars show pathogen colonization in mixed CPcLgene + P biofilms with commensal overexpressing potential colonization resistance genes. Genes deleted or overexpressed are indicated under the bars. Results are averages of at least 12 replicates ± standard deviation of the mean. The extent of colonization in CΔgene + P mixed biofilm was significantly different from that of wild-type C+P biofilm p<0.05; asterisks indicate significant difference between extent of colonization in over-expressed and deletion mutants, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646849&req=5

pone-0061628-g002: Constitutive expression of potential colonization resistance genes.Estimate of E. coli 55989a (P) colonization in mixed biofilms with wild-type E. coli MG1655 F′ (C), corresponding deletion mutants (MG1655Δgene: C*) or overexpressed (MG1655PcLgene: C+) derivative strains. Results are represented as ratio of colonization l of mutant mixed biofilms to wild-type mixed C+P biofilms. Black bar represents wild-type colonization in C+P mixed biofilms arbitrarily set to one. White bars represent colonization of pathogen in mixed CΔgene + P biofilms. Stripped bars show pathogen colonization in mixed CPcLgene + P biofilms with commensal overexpressing potential colonization resistance genes. Genes deleted or overexpressed are indicated under the bars. Results are averages of at least 12 replicates ± standard deviation of the mean. The extent of colonization in CΔgene + P mixed biofilm was significantly different from that of wild-type C+P biofilm p<0.05; asterisks indicate significant difference between extent of colonization in over-expressed and deletion mutants, p<0.05.
Mentions: We then used a previously described plasmid-free expression strategy and placed a constitutive promoter in front of the yiaF, stfE, yliE and ypjC genes directly on the MG1655 F′ chromosome [34], [40]. This insertion had no effect on growth or biofilm formation for yiaF, stfE, yliE and ypjC mutant strains (data not shown). However, constitutive expression of rcsA (MG1655PcLrbs-rcsA F′) led to mucoidy and the inability to form biofilm (data not shown), and this mutant was not further analyzed. The colonization phenotype of the 4 remaining mutants was evaluated and compared to that of the corresponding deletion mutants. While constitutive expression of ypjC and stfE did not lead to significant 55989a colonization changes, yiaF and yliE constitutive expression in MG1655PcLrbs-yiaF F′ and MG1655PcLrbs-yliE F′, respectively, significantly reduced the ability of E. coli pathogen 55989a to colonize the resulting C+P mixed biofilm compared to colonization of the corresponding MG1655 F′ deletion mutants (Fig. 2).

Bottom Line: We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm.We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut.This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Génétique des Biofilms, Département de Microbiologie, Paris, France.

ABSTRACT
Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

Show MeSH
Related in: MedlinePlus