Supplemental Information. Host-Microbiota Interactions in the Pathogenesis. of Antibiotic-Associated Diseases

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1 Cell Reports, Volume Supplemental Information Host-Microbiota Interactions in the Pathogenesis of -Associated Diseases Joshua S. Lichtman, Jessica A. Ferreyra, Katharine M. Ng, Samuel A. Smits, Justin L. Sonnenburg, and Joshua E. Elias

2 A Treatment Salmonella Infection Day: - -Salmonella - +Salmonella n=/group No Pathogen Low Infection + -Salmonella Peak of Infection +Salmonella B Cocktail Treatment C. difficile Infection Fecal Transplant Day: - -C. diff -Clind -Ab cocktail +C. diff n=/group No Pathogen -FT Pathogen below detection Low Infection +Ab cocktail -C. diff +FT Peak of Infection +Clind +C. diff -FT +FT Figure S. -associated Experimental Design,Related to Experimental Procedures. (a) In the Salmonella model, conventionally-raised Swiss Webster mice (n=/group) were treated with mg -hours prior to infection with 8 CFUs of Salmonella typhimurium. Mice were followed for s and fecal pellets were collected prior to antibiotic treatment, prior to infection and and s post-infection. (b) In the C. difficile model, conventional mice were treated with an antibiotic cocktail in drinking water for s, placed on regular drinking water for s and finally given a single dose of mg clindamycin -hours prior to infection with 8 CFUs C. difficile. -s post-infection, mice that received antibiotics (with or without pathogen) received a fecal transplant from the conventional mice. All mice were followed for -s post-infection.

3 A Principal Component # (.%) C Pre-treatment Days post-treatment Treated (Day ) Renin- Kallikrein -related peptidase b7 Major urinary protein Heat shock 7 kda protein B Alpha-defensin-related sequence Transthyretin Serotransferrin Alcohol dehydrogenase Malate dehydrogenase Malate dehydrogenase Heat shock cognate 7 kda protein Calmodulin Cofilin- Selenium-binding protein Cytochrome c Myosin-Ia Destrin Ahnak protein (Fragment) Na+/K+-transporting ATPase subunit alpha- Myosin-VIIb Adseverin Kallikrein related-peptidase Chymotrypsin-like elastase family member A Carboxypeptidase A Elastase B.... Principal Component # (.%) EGFR kinase substrate 8-like protein = MCG8 Hemoglobin subunit beta- UDP-glucose -dehydrogenase Cytochrome P C Ester hydrolase Corf homolog D-dopachrome decarboxylase Medium-chain specific acyl-coa dehydrogenase Oncomodulin Myosin regulatory light chain Ribosome-releasing factor Colipase Phospholipase A Chymotrypsinogen B Pantetheinase Protocadherin E D Pre-treatment Treated (Day ) Days post-treatment Dipeptidyl peptidase Myosin-Ia Histone HB type -F/J/L Superoxide dismutase [Mn] Alcohol dehydrogenase class- Annexin A Tropomyosin alpha- chain Histone H. Prolactin-inducible protein homolog Chymotrypsinogen B Serpin B Annexin A Cytosol aminopeptidase Fumarylacetoacetate hydrolase domain-containing protein Phospholipase A Putative phospholipase B-like Amiloride-sensitive amine oxidase [copper-containing] Cytosolic non-specific dipeptidase Adenosine deaminase Cytochrome c Alpha-actinin- Myosin-9 Hydroxymethylglutaryl-CoA synthase Murinoglobulin- Cadherin-related family member Ig heavy chain V region M Igh protein Chymotrypsin-like elastase family member A Transthyretin Carcinoembryonic antigen-related cell adhesion molecule Igk protein Igh protein (Fragment) Novel protein (ERik) Na(+)/H(+) exchange regulatory cofactor NHE-RF Thioredoxin Mucb protein -mercaptopyruvate sulfurtransferase Biliary glycoprotein Carboxylesterase /7 // / Figure S. Treatment Causes Perturbations to Microbiota Composition and Host Proteome, Related to Figure. PCA analysis of the microbiota upon treatment with (a) and (b), compared to untreated mice over the s after antibiotic treatment. (c,d) Cluster analyses of proteins significantly changed in expression (FDR<., > ln fold change) when comparing (c) clindamycin or (d) streptomycin treated mice - after treatment with the same mice prior treatment. Each column represents a single mouse. Red protein names indicates their significant regulation in both treatment conditions. (e) Venn Diagram comparing the proteins identified in the above analysis. Green= up-regulated in antibiotic-treated condition, Blue= down-regulated in the antibiotic-treated condition, black= up-regulated in clindamycin treatment but down-regulated in streptomycin treatment.

4 A C D.... Relative Abundance of C. diff DNA Principal Component # (.%) 8 Day (Infection on Day ) Conven onal + Salmonella Conven onal + Salmonella + C. diff + C. diff - - (Day ) Principal Component # (.%) + Salmonella (Day ) B Relative Abundance of Salmonella DNA Serum albumin Granulins Actin Annexin A Intelectin-a Glyceraldehyde--phosphate dehydrogenase Cytosol aminopeptidase Neprilysin Zymogen granule membrane protein Cubilin Calmodulin Xaa-Pro dipeptidase Mucin- 78 kda glucose-regulated protein Calcium-activated chloride channel regulator Neutral ceramidase Galectin--binding protein Plastin- Villin- Mucin glycoprotein MUC (Fragment) Regenerating islet-derived protein -gamma Alpha--macroglobulin Chymotrypsin-like elastase family member A Aminopeptidase N Carboxypeptidase B (Tissue) Ela protein (Fragment) E F Relative Spectral Counts Relative Spectral Counts G Relative Spectral Counts Day (Infection on Day ) Serotransferrin Day Complement C Day mice sacrificed Regenerating islet-derived protein -gamma + Salmonella + Salmonella s + C. diff s + C. diff s + C. diff Day Figure S. Host-Microbiota Kinetics During Pathogen Infection, Related to Figure. (a,b) Pathogen load was measured using qpcr assays for (a) C. difficile and (b) Salmonella in mice treated with antibiotics or vehicle controls (mean +/- SEM). (c) PCA of the microbial community composition was conducted on all mice in the Salmonella experiment, s post-infection. (d) Cluster analysis of the proteins significantly (FDR<., ln fold change>) regulated in conventional mice infected with Salmonella -s post-infection. (e-g) From mice in the C. difficile experiment, normalized spectral counts were plotted over time for the innate-immune proteins (e) Serotransferrin, (f) Complement C, and (g) the anti-microbial protein regenerating islet-derived protein -gamma (REG γ) (mean +/- SEM).

5 Unclassified Bacteroidales Bacteroidales Bacteroidaceae Unclassified Clostridiales Clostridiales Ruminococcaceae Enterobacteriales Enterobacteriaceae (relative to ) C. difficile (relative to ) FT (relative to ) FT + C. difficile (relative to ) Figure S. Recovery Dynamics of Individual OTUs Over Day Fecal Transplant Comparison, Related to Figure. Five of the six taxa described in Figure C are represented, as distinct plots for each of the four conditions represented in Figure. As described in Figure C, each colored line represents significant fold changes (natural log, LN) of a single OTU with respect to the same mouse s OUT levels at zero. Black lines line represents the median fold change of OTUs that significantly deviated from their zero levels. No significantly changed OTUs were found for the taxon Lactobacilliales Enterococcaceae (not shown). Numerical fold change values for all taxa can be found in Table S.

6 OTU (Bacteroides) RelativeAbundance s s + FT - 7 Day (Infection on Day ) RelativeAbundance + C. diff + FT - 7 Day (Infection on Day ) Figure S. C. difficile Infection Prevents the Recovery of a Commensal Microbe, Related to Figure. Relative abundance of OTU, a member of the Bacteroides (mean +/- SEM).

7 Table S. Treatment Perturbs the Gastrointestinal Microbiota, Related to Figure. Fold-change for all significantly-different OTUs and s post-antibiotic treatment as measured by DESeq (p<., > ln fold-change). Table S. Salmonella Infection Perturbs the Microbiota in an -Dependent Manner, Related to Figure. Fold change for all significantly-different OTUs on the of Salmonella infection in conventional and streptomycin treated mice, and for the following s, as measured by DESeq (p<., > ln fold-change). Table S. Host Proteins Regulated During -Associated Salmonella Infection, Related to Figure. The fold-change for all significantly-different host proteins when antibiotic-treated, Salmonella infected mice are compared to conventional controls as measured by QSpec (p<., > ln fold-change). Table S. Host Proteins Regulated During DSS Colitis, Related to Figure. The fold-change for all significantly-different host proteins when DSS-treated mice are compared to the same mice prior to treatment, as measured by QSpec (p<., > ln fold-change). Table S. Recovery of the Microbiota After, C. difficile Infection and Fecal Transplant, Related to Figure. The significantly-different OTUs from the groups of mice that received clindamyicn treatment throughout the last s of the experiment as measured by DESeq (p<., > ln fold-change). Table S. Protein abundance (spectral counts) corresponding with all mice and time points, Related to Figures -. Table S7. Taxon abundance (normalized S rdna sequence reads) corresponding with all mice and time points, Related to Figures, and.