Resources to Facilitate Use of the Altered Schaedler Flora (ASF) Mouse Model to Study Microbiome Function

Abstract

Animals colonized with a defined microbiota represent useful experimental systems to investigate microbiome function. The altered Schaedler flora (ASF) represents a consortium of eight murine bacterial species that have been used for more than 4 decades where the study of mice with a reduced microbiota is desired. In contrast to germ-free mice, or mice colonized with only one or two species, ASF mice show the normal gut structure and immune system development. To further expand the utility of the ASF, we have developed technical and bioinformatic resources to enable a systems-based analysis of microbiome function using this model. Here, we highlighted four distinct applications of these resources that enable and improve (i) measurements of the abundance of each ASF member by quantitative PCR; (ii) exploration and comparative analysis of ASF genomes and the metabolic pathways they encode that comprise the entire gut microbiome; (iii) global transcriptional profiling to identify genes whose expression responds to environmental changes within the gut; and (iv) discovery of genetic changes resulting from the evolutionary adaptation of the microbiota. These resources were designed to be accessible to a broad community of researchers that, in combination with conventionally-reared mice (i.e., with complex microbiome), should contribute to our understanding of microbiome structure and function. IMPORTANCE Improved experimental systems are needed to advance our understanding of how the gut microbiome influences processes of the mammalian host as well as microbial community structure and function. An approach that is receiving considerable attention is the use of animal models that harbor a stable microbiota of known composition, i.e., defined microbiota, which enables control over an otherwise highly complex and variable feature of mammalian biology. The altered Schaedler flora (ASF) consortium is a well-established defined microbiota model, where mice are stably colonized with 8 distinct murine bacterial species. To take better advantage of the ASF, we established new experimental and bioinformatics resources for researchers to make better use of this model as an experimental system to study microbiome function.

Keywords: ASF; Schaedler; gnotobiotic mice; microbiome function; microbiota evolution; qPCR.

FIG 1

Identification and comparison of bile acid deconjugation pathways among ASF members by PTools software. (A) Five known pathways for metabolism of primary bile acids by bacterial deconjugation reactions. Specific deconjugation pathways are indicated by the glyphs below each group of conversions reactions. The numbers correspond to the specific enzymatic reactions (RXN) shown in the table. (B) The table shows the presence or absence of bile acid deconjugation pathways within each ASF member and was modified from the output of PTools. Green lines in the glyphs indicate that the pathway is present, while black lines indicate the pathway is not found. Orange lines indicate that reaction is unique to bile acid deconjugation compared to all other pathways in BioCyc. Inspection of the table revealed that choloylglycine hydrolase activity was predicted in ASF356, 360, 361, and 519, which was specific for the primary bile acids in pathways 3 and 5. No evidence of chenodeoxycholyltaurine hydrolase activity (pathways 1, 2, and 4) was found. In addition, no evidence of bile acid deconjugation activity was found in the genomes of ASF457, 492, 500, and 502. For the remaining ASF members, the locus tag and name of the enzymes they encode are shown. For the gene products listed as hypothetical or putative, the protein sequence was used to identify homologous proteins using BLASTp (NCBI) and including ^aASF356 (locus C820_000635), identical to choloylglycine hydrolase in Clostridium sp. Isolate MD294; ^bASF519 (locus C825_000821), identical to a linear amide C-N hydrolase in another Parabacteroides goldsteinii isolate; ^cASF519 (yxeI, locus C825_001702), highly similar to a choloylglycine hydrolase from Bacillus mohavensis.

FIG 2

Abundance of the ASF in three strains of gnotobiotic mice. The absolute (A and B) and relative abundance (C) of the ASF were compared using different approaches. (A) The abundance of the ASF was compared between different mouse strains using groEL qPCR primers. (B) The abundance of ASF361, ASF457, and ASF519 was compared using groEL and 16S rRNA gene qPCR primers. (C) Relative abundance of the ASF using qPCR, 16S rRNA gene amplicon (16S) and metagenomic sequences from different mouse backgrounds: C3H/HeNTac (C3H) (n = 2 [qPCR], n = 13 [16S], n = 2 [metagenomics]); 129S6/SvEv wild type (WT) (n = 2 [qPCR], n = 7 [16S]) and 129S6/SvEv IL-10^−/− (KO) (n = 2 [qPCR], n = 5 [16S]).

FIG 3

Treatment of mice with azoxymethane (AOM) plus dextran sulfate sodium (DSS) results in gene expression changes in the ASF. (A) Genes with the highest positive fold change were found in individual ASF members. (B) An example of the Pathway Tools ‘Omics Dashboard output used to assess the impact of AOM+DSS exposure on cellular functions of M. schaedleri (ASF457). Insert compared pathways involved in detoxification processes at higher resolution. Similar comparisons can be conducted on each ASF member using Pathway Tools. The ‘Omics Dashboard also allowed for more detailed visualization using the associated toolboxes at the bottom of each section.

FIG 4

Genetic map of the M. schaedleri (ASF457) chromosome in the region encoding components of the type VI secretion system. (A) The location of the 3 homologous vgrG genes is shown, along with adjacent open reading frames (arrows). The location of the deletion mutation is shown by the square bracket. (B) The same region was identified from metagenomic sequences. The DNA sequence at the junction of the deletion is shown along with the location of PCR primers used to identify the deletion.