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SYNTHESIS OF THE INTERSPECIES SIGNALING MOLECULE AUTOINDUCER-2 PLAYS AN IMPORTANT ROLE IN THE ANTI-MICROBIAL EFFECT OF BIFIDOBACTERIA

ABSTRACT: In the present study we show that a functional luxS gene is present in all members of the genus Bifidobacterium and that AI-2 production by Bifidobacterium breve UCC2003 is necessary to confer protection against Salmonella infection in Caenorhabditis elegans. Mutants carrying a disrupted luxS gene conferred less protection than the WT, whereas administration of a complemented strain, a LuxS-overexpression strain and the WT resulted in similar survival. Transcriptome data obtained for both the WT and the luxS-insertion mutant grown in vitro suggest that LuxS has a metabolic role that impacts on iron metabolism. In line with this, we demonstrated that the luxS mutant is significantly more susceptible to the ferrous ion chelator 2,2-dipyridyl than the WT strain. Furthermore, qRT-PCR shows that the iron regulated genes are upregulated under in vivo conditions, while we also show that LuxS is required for colonization of BALB/c mice. Altogether, our findings provide detailed insights into the molecular mechanism underlying gut pathogen protection conferred by bifidobacteria.

ORGANISM(S): Bifidobacterium breve UCC2003

PROVIDER: GSE49880 | GEO | 2015/12/31

SECONDARY ACCESSION(S): PRJNA215755

REPOSITORIES: GEO

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Project description:In the present study we show that a functional luxS gene is present in all members of the genus Bifidobacterium and that AI-2 production by Bifidobacterium breve UCC2003 is necessary to confer protection against Salmonella infection in Caenorhabditis elegans. Mutants carrying a disrupted luxS gene conferred less protection than the WT, whereas administration of a complemented strain, a LuxS-overexpression strain and the WT resulted in similar survival. Transcriptome data obtained for both the WT and the luxS-insertion mutant grown in vitro suggest that LuxS has a metabolic role that impacts on iron metabolism. In line with this, we demonstrated that the luxS mutant is significantly more susceptible to the ferrous ion chelator 2,2-dipyridyl than the WT strain. Furthermore, qRT-PCR shows that the iron regulated genes are upregulated under in vivo conditions, while we also show that LuxS is required for colonization of BALB/c mice. Altogether, our findings provide detailed insights into the molecular mechanism underlying gut pathogen protection conferred by bifidobacteria. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilentâs standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

Project description:In a transcriptome based trail, we figured out that the deletion of luxS has a massive influence to cell growth and metabolism of Streptococcus sanguinis SK36. The biofilm defective luxS deletion mutant was comlemented by transgenic sahH from Pseudomonas aeruginosa. Thus 209 of 216 influenced genes of the luxS mutant compared to the isogenic wildype were restored in their expression (fold change of n ≥ 3; p ≤ 0.05), including genes involved in cell division processes, stress response and catabolite control. Phenotypically, the reduced biofilm depth of S. sanguinis SR DELTAluxS was elevated to wild type level by the complementation with sahH. Neither the addition of physiological concentrated artificial autoinducer 2 (AI-2) to the culture of S. sanguinis SR DELTAluxS nor the presence of the wild type strain in a trans-well assay had a cumulative effect on biofilm thickness of the luxS mutant. Furthermore, we identified 9 genes in the sahH complemented luxS mutant, which were regulated directly by AI-2 (fold change ≥ 3; p ≤ 0.05), amongst them genes involved in competence development. So we concluded that AI-2 has no influence on biofilm growth, but regulative functions in competence development in S. sanguinis. Further, we figured out the suitability of transgenic sahH for the complementation of the interrupted Pfs/LuxS pathway without restoration of AI-2 release acquiring an essential tool for further investigations on AI-2. Aim: Is the presence of AI-2 necessary for the biofilm formation of S. sanguinis SK36? What is the impact of a deletion of luxS, is a disrupted activated methionine cycle the reason for the hampered biofilm depth causes by its inactivation? Materials and Methods: S. sanguinis SK36, S. sanguinis SR ΔluxS, and S. sanguinis SR ΔluxS/sahH were grown in CDM/sucrose for 24 h anaerobically; biofilm depth was measured by safranine and crystal violet stain. Biofilm morphology was investigated by scanning electron microscopy. The AI-2 release was monitored hourly to identify the time period of its release. For transcriptome analysis total RNA was isolated after 8 hours of growth and used for microarray analysis.

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SYNTHESIS OF THE INTERSPECIES SIGNALING MOLECULE AUTOINDUCER-2 PLAYS AN IMPORTANT ROLE IN THE ANTI-MICROBIAL EFFECT OF BIFIDOBACTERIA

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