Research ArticleMalnutrition

Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy

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Science Translational Medicine  25 Feb 2015:
Vol. 7, Issue 276, pp. 276ra24
DOI: 10.1126/scitranslmed.aaa4877
  • Fig. 1. IgA responses in gnotobiotic mice colonized with the fecal microbiota of twins discordant for kwashiorkor.

    Diet- and microbiota-associated differences in IgA responses to bacterial taxa present in gnotobiotic mice containing transplanted microbiota from kwashiorkor or healthy co-twins from discordant pair 57. Separate groups of mice received fecal microbiota from either the kwashiorkor (K) or healthy (H) co-twin and were fed an irradiated (sterile) prototypic Malawian (M) diet (KM or HM groups) or a control nutrient-sufficient standard (S) mouse chow (KS and HS groups). Fecal samples collected from recipient mice 13 to 16 days after gavage of the human donor microbiota were analyzed by BugFACS. Results shown are from two independent experiments. (A) Enterobacteriaceae were significantly enriched in the IgA+ fraction prepared from the fecal microbiota of KM mice compared to all other groups of animals, indicating the microbiota and diet specificity of the gut mucosal immune response. Each data point represents a fecal microbiota sample from a different animal. (B) Mice colonized with the microbiota from the healthy co-twin had an IgA response to Verrucomicrobiaceae that was significantly greater than the responses of mice harboring microbiota from the sibling with kwashiorkor. **P < 0.01; ***P < 0.001; ****P < 0.0001 (Wilcoxon rank sum test). (C) Bubble plot depicting IgA responses (defined by the IgA index) to different family-level bacterial taxa. Each column represents a different group of gnotobiotic mice. Each row shows the relative enrichment of a given family-level taxon in the IgA+ or IgA fraction. The color of the circle represents the direction of enrichment, with darker colors indicating greater significance as determined by paired Wilcoxon test (threshold for significantly enriched, P < 0.05). The diameter of a given circle represents the average magnitude of enrichment for a given taxon in the fecal microbiota of members of a given treatment group. n, number of gnotobiotic mice analyzed per treatment group (see table S2 for IgA indices for members of each family-level taxon shown). n.s., not significant. Gray circles indicate that a taxon was not observed within a given treatment group.

  • Fig. 2. Bacterial targets of IgA responses to kwashiorkor and healthy co-twin microbiota introduced into germ-free mice.

    Adult germ-free C57BL/6J mice were fed an irradiated Malawian diet starting 1 week before gavage with IgA+ fractions. These fractions were purified from fecal microbiota obtained from KM or HM mice 42 days after they had been colonized with the respective co-twin’s microbiota. After gavage, the recipient mice were maintained on the Malawian diet. (A) KMIgA+ mice (n = 20) experienced significantly greater mortality than HMIgA+ mice (n = 15). Mice that received an equivalent number of cells from IgA+ consortia purified from HM and KM mouse fecal microbiota did not exhibit mortality during the course of the experiment (MixIgA+, n = 10). *P < 0.05; **P < 0.01, compared to the KMIgA+ group (Fisher’s exact test). Results represent data from two independent experiments. (B) Impact of diet. KMIgA+ mice fed a Malawian diet lost more weight over a 2-week period after colonization than did animals colonized with the same IgA+ consortium but fed a standard nutrient-sufficient mouse chow. Mice receiving the IgA+ consortium purified from the fecal microbiota of mice harboring the same family 57 kwashiorkor donor microbiota but fed a standard mouse chow (KS IgA+ mice) lost less weight than did KMIgA+ mice, regardless of whether they were fed the Malawian diet or a standard mouse chow. *P < 0.05; **P < 0.01; ***P < 0.001 (Wilcoxon rank sum test). (C) C. scindens was present in the fecal microbiota of HM, HMIgA+, and MixIgA+ mice but was not detectable in the microbiota of KM or KMIgA+ animals. *P < 0.05; ***P < 0.001; ****P < 0.0001 (Wilcoxon rank sum test). (D) Experimental design of a follow-up experiment where three groups of adult germ-free male mice were gavaged with an IgA+ consortium purified by BugFACS from the fecal microbiota of surviving KMIgA+ mice. All recipients [second-generation (F2) KMF2IgA+ mice] were fed the Malawian diet. The first group of KMF2IgA+ mice received no intervention (n = 10). Another group received an equal mixture of live C. scindens and A. muciniphila by gavage 24 hours before introduction of the IgA+ consortium purified from KMIgA+ mice (CsAm + KMF2IgA+, n = 15). A third group was gavaged with heat-killed C. scindens and A. muciniphila 24 hours before introduction of the IgA+ consortium (heat-killed CsAm + KMF2IgA+, n = 5). (E) CsAm + KMF2IgA+ mice exhibited significantly reduced mortality compared to either KMF2IgA+ or heat-killed CsAm + KMF2IgA+ animals. **P < 0.01 (Fisher’s exact test). (F) Effects of colonizing germ-free mice fed a Malawian diet with different components of the 11 OTU culture collection, generated from KMF2IgA+ microbiota, on weight. Data for individual mice in each treatment group are plotted. ***P < 0.001; ****P < 0.0001 (Wilcoxon rank sum test).

  • Fig. 3. Identifying bacterial strains that transmit gut barrier disruption phenotypes.

    Adult germ-free mice consuming the prototypic Malawian diet were gavaged with all 11 OTUs contained in the clonally arrayed culture collection generated from the cecal microbiota of KMF2IgA+ mice, or two subsets of the culture collection: a consortium of the five strains belonging to Enterobacteriaceae (E. coli, K. variicola, and Citrobacter amalonaticus) and Enterococcus (E. hirae and E. casseliflavus) or a consortium of eight strains that included all but the three strains of Enterobacteriaceae in the collection (see table S3 for details about the genome sequences of these organisms, including their virulence factor content). All animals were sacrificed 2 days after gavage, and hematoxylin and eosin–stained sections of their proximal colons were prepared. (A) Colonization with the 11 OTU consortium produced generalized disruption of the colonic epithelium with marked loss of crypts. (B) The epithelium and crypt numbers were preserved in mice harboring the five-strain consortium. (C) The eight-strain consortium lacking members of Enterobacteriaceae did not produce the epithelial disruption seen with the entire 11-strain consortium, and crypts were largely preserved. However, there was an associated neutrophil infiltrate in the lamina propria (highlighted in inset). (D) Quantification of crypt number per unit area of the colonic epithelium. *P < 0.05; **P < 0.01 (Wilcoxon rank sum test).

  • Fig. 4. Enterobacteriaceae are targeted by the gut mucosal IgA response in children from the Malawian twin study.

    (A) IgA responses were defined by BugFACS of fecal samples obtained from 11 twin pairs discordant for kwashiorkor. Data from five time points are shown. The first column represents samples taken 1.2 ± 0.6 months before diagnosis of kwashiorkor. The second column represents samples taken at the time of diagnosis. The third and fourth columns are samples taken 2 and 4 weeks after initiation of treatment with RUTF, whereas the fifth column represents fecal microbiota characterized 1 month after the completion of RUTF therapy. Data represent mean values for the indicated number (n) of kwashiorkor co-twins and healthy co-twins whose fecal samples were available, and are presented in the form of a bubble plot. See tables S1 and S4 for clinical characteristics and details of the data sets, including IgA indices for individual taxa identified as present within each family-level taxonomic group for each individual fecal sample analyzed. (B) At the time of diagnosis, the IgA index for Enterobacteriaceae was significantly higher in co-twins with kwashiorkor in discordant pairs than in twin pairs concordant for healthy status (data from twins concordant for healthy status represent the averaged IgA indices of an individual’s fecal specimens obtained between 6 and 24 months of age to allow for comparison with discordant twins of varying ages at the time of diagnosis). Purple and green circles highlight IgA indices for co-twins in discordant pairs 46 and 80, who were used for microbial adoptive transfer experiments [see (D) and (E)]. **P < 0.01 (Wilcoxon rank sum test). (C) Treatment of kwashiorkor co-twins in all 11 discordant pairs shown in (A and B) with RUTF resulted in a significant decrease in the IgA index score for Enterobacteriaceae. *P < 0.05 (Wilcoxon rank sum test). Data represent the average IgA index scores for samples obtained 2 and 4 weeks after initiation of RUTF treatment. Mean values ± SEM are plotted. (D) Germ-free mice colonized with a BugFACS-purified IgA+ consortium from the kwashiorkor child in twin pair 46 lost more weight than mice colonized with either the IgA+ consortium purified from the fecal microbiota of his healthy co-twin or a mixture of the two IgA+ populations. **P < 0.01 (Wilcoxon rank sum test). (E) Colonization of germ-free mice with KwashIgA+, HealthyIgA+, or MixIgA+ consortia prepared from discordant twin pair 80, whose members had similar IgA index values for Enterobacteriaceae [see (B)], did not exhibit significant differences in weight loss (n = 5 to 7 mice per treatment group). All mice were fed the Malawian diet starting 1 week before gavage with the IgA+ consortia. Body weights at the time of sacrifice 13 days after gavage were used to plot the data shown (each mouse represented by a circle).

  • Fig. 5. Relationships among IgA indices, enteropathogen burden, and nutritional status in 18-month-old Malawian children from the LCNI-5 cohort.

    (A) IgA indices for Enterobacteriaceae were significantly higher in children that harbored EPEC and EAEC in their microbiota. Each circle represents results from an individual child. **P < 0.01 (Wilcoxon rank sum test). (B) ROC curves for detection of EPEC (eae) and/or EAEC (aggR) using either Enterobacteriaceae relative abundance (purple, defined by 16S rRNA sequencing) or Enterobacteriaceae IgA index (orange). Samples were excluded where Enterobacteriaceae were not detected by 16S rRNA sequencing. The presence of eae or aggR was significantly correlated to the IgA index (P < 0.01; binomial logistic regression) but not to the relative abundance. (C) Eighteen-month-old children from the LCNI-5 cohort with IgA indices > 0.25 for Enterobacteriaceae had lower WHZ scores than did children with indices < 0.25. **P < 0.01 (Welch’s t test). (D) Feature importance scores of bacterial taxa that are predictive of LAZ scores were generated by training a sparse Random Forests model using age and genus-level IgA index data from 134 fecal samples collected from the 11 kwashiorkor discordant twin pairs and the 8 concordant healthy pairs enrolled in the Malawi Twin Study. To build the model, we included genus-level taxa (features) that had an IgA index value greater than 0.05 or less than −0.05 in 30% of all fecal samples (to remove genera that were only rarely seen and/or had very little enrichment in either the IgA+ or IgA fraction). The IgA indices for the 25 taxa that satisfied this criterion were regressed against LAZ, and feature importance scores for each genus-level taxon were defined (mean values ± SD are shown). Shown are the nine genus-level taxa with mean importance scores greater than 1.5% that were incorporated into a 10-feature sparse model, which also included the chronological age of a child. The plot in the inset shows that application of this model to 165 fecal samples collected from 6- and 18-month-old singleton children in the LCNI-5 study predicted LAZ scores that correlated significantly with their actual LAZ measurements (Spearman’s ρ = 0.2, P = 0.009).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/276/276ra24/DC1

    Fig. S1. Characterization of gnotobiotic mice receiving intact uncultured fecal microbiota from discordant twins and the experimental design for subsequent transplantation of IgA+ consortia purified from their fecal microbiota into germ-free recipients.

    Fig. S2. Validation of BugFACS using an artificial two-member bacterial community.

    Fig. S3. Control experiments validating the specificity of BugFACS.

    Fig. S4. Combining HMIgA+ and KMIgA+ consortia attenuates early weight loss in recipient gnotobiotic animals.

    Fig. S5. Serum cytokine profiles in KMIgA+ animals and a comparison of immunophenotypes in recipients of KMIgA+, HMIgA+, and MixIgA+ consortia.

    Fig. S6. KMF2IgA+ microbiota transmits a colonic epithelial discohesion phenotype to recipient gnotobiotic mice.

    Fig. S7. IgA-targeted bacterial taxa in the kwashiorkor microbiota disrupt the small intestinal epithelial barrier in recipient gnotobiotic mice.

    Fig. S8. Representation of bacterial taxa within the gut microbiota of gnotobiotic mice colonized with IgA+ consortia.

    Fig. S9. Heatmap of bacterial family-level taxa represented in the fecal microbiota of gnotobiotic mice sampled 12 days after receiving IgA+ consortia from discordant twin pair 46 or 80.

    Table S1. Clinical characteristics of Malawian twin microbiota donors.

    Table S2. IgA indices determined from BugFACS analysis of fecal samples obtained from mice colonized with the fecal microbiota of co-twins discordant for kwashiorkor (pair 57).

    Table S3. Characteristics of the genome sequences and content of virulence factors of Enterobacteriaceae isolates obtained from in vitro culture of IgA+ consortia or from spleens of mice colonized with KMF2IgA+ cecal microbiota (originating from donor 57A).

    Table S4. IgA indices and relative abundances of family- and genus-level bacterial taxa in two cohorts of Malawian infants.

    Table S5. Clinical characteristics of individuals from the LCNI-5.

    Table S6. Multiplex quantitative PCR analysis of enteropathogen burden in members of the LCNI-5 cohort.

    Table S7. Subgroup analysis of LCNI-5 cohort.

  • Supplementary Material for:

    Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy

    Andrew L. Kau, Joseph D. Planer, Jie Liu, Sindhuja Rao, Tanya Yatsunenko, Indi Trehan, Mark J. Manary, Ta-Chiang Liu, Thaddeus S. Stappenbeck, Kenneth M. Maleta, Per Ashorn, Kathryn G. Dewey, Eric R. Houpt, Chyi-Song Hsieh, Jeffrey I. Gordon*

    *Corresponding author. E-mail: jgordon@wustl.edu

    Published 25 February 2015, Sci. Transl. Med. 7, 276ra24 (2015)
    DOI: 10.1126/scitranslmed.aaa4877

    This PDF file includes:

    • Fig. S1. Characterization of gnotobiotic mice receiving intact uncultured fecal microbiota from discordant twins and the experimental design for subsequent
      transplantation of IgA+ consortia purified from their fecal microbiota into germ-free recipients.
    • Fig. S2. Validation of BugFACS using an artificial two-member bacterial community.
    • Fig. S3. Control experiments validating the specificity of BugFACS.
    • Fig. S4. Combining HMIgA+ and KMIgA+ consortia attenuates early weight loss in recipient gnotobiotic animals.
    • Fig. S5. Serum cytokine profiles in KMIgA+ animals and a comparison of immunophenotypes in recipients of KMIgA+, HMIgA+, and MixIgA+ consortia.
    • Fig. S6. KMF2IgA+ microbiota transmits a colonic epithelial discohesion phenotype to recipient gnotobiotic mice.
    • Fig. S7. IgA-targeted bacterial taxa in the kwashiorkor microbiota disrupt the small intestinal epithelial barrier in recipient gnotobiotic mice.
    • Fig. S8. Representation of bacterial taxa within the gut microbiota of gnotobiotic mice colonized with IgA+ consortia.
    • Fig. S9. Heatmap of bacterial family-level taxa represented in the fecal microbiota of gnotobiotic mice sampled 12 days after receiving IgA+ consortia from
      discordant twin pair 46 or 80.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Clinical characteristics of Malawian twin microbiota donors.
    • Table S2 (Microsoft Excel format). IgA indices determined from BugFACS analysis of fecal samples obtained from mice colonized with the fecal microbiota
    • of co-twins discordant for kwashiorkor (pair 57).
    • Table S3 (Microsoft Excel format). Characteristics of the genome sequences and content of virulence factors of Enterobacteriaceae isolates obtained from in vitro culture of IgA+ consortia or from spleens of mice colonized with KMF2IgA+ cecal microbiota (originating from donor 57A).
    • Table S4 (Microsoft Excel format). IgA indices and relative abundances of family- and genus-level bacterial taxa in two cohorts of Malawian infants.
    • Table S5 (Microsoft Excel format). Clinical characteristics of individuals from the LCNI-5.
    • Table S6 (Microsoft Excel format). Multiplex quantitative PCR analysis of enteropathogen burden in members of the LCNI-5 cohort.
    • Table S7 (Microsoft Excel format). Subgroup analysis of LCNI-5 cohort.

    [Download Tables S1 to S7]

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