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Monitoring Perinatal Gut Microbiota in Mouse Models by Mass Spectrometry Approaches: Parental Genetic Background and Breastfeeding Effects

View Article: PubMed Central - PubMed

ABSTRACT

At birth, contact with external stimuli, such as nutrients derived from food, is necessary to modulate the symbiotic balance between commensal and pathogenic bacteria, protect against bacterial dysbiosis, and initiate the development of the mucosal immune response. Among a variety of different feeding patterns, breastfeeding represents the best modality. In fact, the capacity of breast milk to modulate the composition of infants’ gut microbiota leads to beneficial effects on their health. In this study, we used newborn mice as a model to evaluate the effect of parental genetic background (i.e., IgA-producing mice and IgA-deficient mice) and feeding modulation (i.e., maternal feeding and cross-feeding) on the onset and shaping of gut microbiota after birth. To investigate these topics, we used either a culturomic approach that employed Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MS), or bottom–up Liquid Chromatography, with subsequent MSMS shotgun metaproteomic analysis that compared and assembled results of the two techniques. We found that the microbial community was enriched by lactic acid bacteria when pups were breastfed by wild-type (WT) mothers, while IgA-deficient milk led to an increase in the opportunistic bacterial pathogen (OBP) population. Cross-feeding results suggested that IgA supplementation promoted the exclusion of some OBPs and the temporary appearance of beneficial species in pups fed by WT foster mothers. Our results show that both techniques yield a picture of microbiota from different angles and with varying depths. In particular, our metaproteomic pipeline was found to be a reliable tool in the description of microbiota. Data from these studies are available via ProteomeXchange, with identifier PXD004033.

No MeSH data available.


Graphical representation of culturomics- and metaproteomics-based pipelines. The scheme outlines the mouse breeding step, the pre-analytical phase and the two analytical methodologies, consisting of culturomics and metaproteomics pipelines.
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Figure 1: Graphical representation of culturomics- and metaproteomics-based pipelines. The scheme outlines the mouse breeding step, the pre-analytical phase and the two analytical methodologies, consisting of culturomics and metaproteomics pipelines.

Mentions: Mouse gut microbiota phylotypes were grouped at different taxonomic levels by applying a modified version of the Excel macro developed by Del Chierico et al. (2014). More specifically, a script in Python Programming Language was created to implement the whole workflow of the original Excel macro, starting from the Mascot “csv” exported files to produce two different representations (i.e., workflows A and B) of the taxonomic distribution of the OTUs. Workflow A was obtained by counting the number of peptides associated with one or more taxa, namely, including peptides present in the same protein hit but associated with different OTUs, or peptides found in different protein hits and associated with the same OTU. Workflow B reported the number of peptides that could be associated with a single OTU only (Figure 1). To let the program work properly, a NCBI “taxon ID” was previously assigned to each NCBI “gi accession number” by a second Python script, then the whole taxonomy lineage was associated with each peptide entry, allowing the application of a counting workflow at every taxonomic level. A threshold value of 10 on the peptide score was set as an input for the script.


Monitoring Perinatal Gut Microbiota in Mouse Models by Mass Spectrometry Approaches: Parental Genetic Background and Breastfeeding Effects
Graphical representation of culturomics- and metaproteomics-based pipelines. The scheme outlines the mouse breeding step, the pre-analytical phase and the two analytical methodologies, consisting of culturomics and metaproteomics pipelines.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036385&req=5

Figure 1: Graphical representation of culturomics- and metaproteomics-based pipelines. The scheme outlines the mouse breeding step, the pre-analytical phase and the two analytical methodologies, consisting of culturomics and metaproteomics pipelines.
Mentions: Mouse gut microbiota phylotypes were grouped at different taxonomic levels by applying a modified version of the Excel macro developed by Del Chierico et al. (2014). More specifically, a script in Python Programming Language was created to implement the whole workflow of the original Excel macro, starting from the Mascot “csv” exported files to produce two different representations (i.e., workflows A and B) of the taxonomic distribution of the OTUs. Workflow A was obtained by counting the number of peptides associated with one or more taxa, namely, including peptides present in the same protein hit but associated with different OTUs, or peptides found in different protein hits and associated with the same OTU. Workflow B reported the number of peptides that could be associated with a single OTU only (Figure 1). To let the program work properly, a NCBI “taxon ID” was previously assigned to each NCBI “gi accession number” by a second Python script, then the whole taxonomy lineage was associated with each peptide entry, allowing the application of a counting workflow at every taxonomic level. A threshold value of 10 on the peptide score was set as an input for the script.

View Article: PubMed Central - PubMed

ABSTRACT

At birth, contact with external stimuli, such as nutrients derived from food, is necessary to modulate the symbiotic balance between commensal and pathogenic bacteria, protect against bacterial dysbiosis, and initiate the development of the mucosal immune response. Among a variety of different feeding patterns, breastfeeding represents the best modality. In fact, the capacity of breast milk to modulate the composition of infants’ gut microbiota leads to beneficial effects on their health. In this study, we used newborn mice as a model to evaluate the effect of parental genetic background (i.e., IgA-producing mice and IgA-deficient mice) and feeding modulation (i.e., maternal feeding and cross-feeding) on the onset and shaping of gut microbiota after birth. To investigate these topics, we used either a culturomic approach that employed Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MS), or bottom–up Liquid Chromatography, with subsequent MSMS shotgun metaproteomic analysis that compared and assembled results of the two techniques. We found that the microbial community was enriched by lactic acid bacteria when pups were breastfed by wild-type (WT) mothers, while IgA-deficient milk led to an increase in the opportunistic bacterial pathogen (OBP) population. Cross-feeding results suggested that IgA supplementation promoted the exclusion of some OBPs and the temporary appearance of beneficial species in pups fed by WT foster mothers. Our results show that both techniques yield a picture of microbiota from different angles and with varying depths. In particular, our metaproteomic pipeline was found to be a reliable tool in the description of microbiota. Data from these studies are available via ProteomeXchange, with identifier PXD004033.

No MeSH data available.