www.sciencemag.org/content/358/6361/359/suppl/DC1

Supplementary Materials for

Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation

Koji Atarashi, Wataru Suda, Chengwei Luo, Takaaki Kawaguchi, Iori Motoo, Seiko Narushima, Yuya Kiguchi, Keiko Yasuma, Eiichiro Watanabe, Takeshi Tanoue, Christoph A. Thaiss, Mayuko Sato, Kiminori Toyooka, Heba S. Said, Hirokazu

Yamagami, Scott A. Rice, Dirk Gevers, Ryan C. Johnson, Julia A. Segre, Kong Chen, Jay K. Kolls, Eran Elinav, Hidetoshi Morita, Ramnik J. Xavier, Masahira Hattori,*

Kenya Honda*

*Corresponding author. Email: m-hattori@aoni.waseda.jp (M.H.); kenya@keio.jp (K.H.)

Published 20 October 2017, Science 358, 359 (2017) DOI: 10.1126/science.aan4526

This PDF file includes:

Materials and Methods Figs. S1 to S12 References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/358/6361/359/suppl/DC1)

Tables S1 to S4

Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and

inflammation

Koji Atarashi, Wataru Suda, Chengwei Luo, Takaaki Kawaguchi, Iori Motoo, Seiko Narushima, Yuya Kiguchi,

Keiko Yasuma, Eiichiro Watanabe, Takeshi Tanoue, Christoph A. Thaiss, Mayuko Sato, Kiminori Toyooka,

Heba S. Said, Hirokazu Yamagami, Scott A. Rice, Dirk Gevers, Ryan C. Johnson, Julia A. Segre, Kong Chen, Jay K. Kolls, Eran Elinav, Hidetoshi Morita, Ramnik J. Xavier, Masahira Hattori, Kenya Honda

Supplementary Materials Materials and methods

Tables S1-S4

Figures S1 to S12

References (34-39)

Materials and Methods

Mice

C57BL/6, BALB/c, and IQI mice maintained under SPF or GF conditions were purchased from Sankyo

Laboratories Japan, SLC Japan, or CLEA Japan. GF and gnotobiotic mice were bred and maintained within the

gnotobiotic facility of Keio University School of Medicine or RIKEN IMS. Il10-/-, Ifngr1-/-, Batf3-/-, Il18-/-, and

Il1r1-/- mice were purchased from the Jackson Laboratories. Myd88-/-, Tlr4-/- and Myd88-/- Trif-/- mice were

purchased from Oriental Bio Service (Japan). All animal experiments were approved by the Keio University

Institutional Animal Care and Use Committee and RIKEN Yokohama Institute.

16S rRNA gene pyrosequencing

The feces from mice were suspended in 20% glycerol/PBS containing 10mM Tris-HCl (pH 8.0) to a final

concentration of 10% (w/v) and stored at -80°C. Frozen sample was thawed and 100 µL of suspensions was

mixed with 900 µL TE10 (10mM Tris-HCl, 10mM EDTA) buffer containing RNase A (final concentration of

100 µg/mL, Invitrogen) and lysozyme (final 3.0 mg/mL, Sigma). The suspension was incubated for 1 h at 37°C

with gentle mixing. Purified achromopeptidase (Wako) was added to a final concentration of 2,000 unit/mL, and

the sample was further incubated for 30 min at 37°C. Then, sodium dodecyl sulfate (final 1%) and proteinase K

(final 1 mg/mL, Nacalai) were added to the suspension and the mixture was incubated for 1 h at 55°C. High-

molecular mass DNA was extracted by phenol:chloroform:isoamyl alcohol (25:24:1), precipitated by isopropanol,

washed with 70% ethanol, and resuspended in 200 µL of TE. PCR was performed using Ex Taq (Takara) and (1)

the 454 primer A [5ʹ-CCATCTCATCCCTGCGTGTCTCCGACTCAG (454 adaptor sequence) + barcode (10

bases) + AGRGTTTGATYMTGGCTCAG-3ʹ (27Fmod)] and (2) the 454 primer B [5ʹ-

2

CCTATCCCCTGTGTGCCTTGGCAGTCTCAG (454 adaptor sequence) + TGCTGCCTCCCGTAGGAGT-3ʹ

(338R)] to the V1–V2 region of the 16S rRNA gene. Amplicons generated from each sample (~330 bp) were

subsequently purified using AMPure XP (Beckman Coulter). DNA was quantified using a Quant-iT Picogreen

dsDNA assay kit (Invitrogen) and a TBS-380 Mini-Fluorometer (Turner Biosystems). Then, the amplified DNA

was used as template for 454 GS Junior (Roche) pyrosequencing using GS Junior Titanium emPCR Kit-Lib-L,

GS Junior Titanium Sequencing Kit, and GS Junior Titanium PicoTiterPlate Kit (all from Roche) according to

the manufacturer’s instructions. Quality filter–passed reads were obtained by removing reads that did not have

both primer sequences, had an average quality value of <25, and were possibly chimeric. Of the filter-passed

reads, 3,000 reads were used after trimming off both primer sequences for each sample and subjected to OTU

analysis with the cutoff similarity of 96% identity. Representative sequences from each OTU were blasted to

databases from the Ribosomal Database Project (RDP) and our genome database constructed from publicly

available genome sequences in NCBI and Human Microbiome Project databases.

Human saliva samples, bacterial culture, and generation of gnotobiotic animals

Human saliva samples were collected at the Hospital of University of the Ryukyus according to the study protocol

approved by the institutional review board as described previously (34). Informed consent was obtained from

each subject. Saliva samples from patients with CD [#1:IBD029, 50-year-old Japanese man, IOIBD score 3

(active-phase); #2:IBD121, 52-year-old Japanese man, IOIBD score 1 (remission-phase)], UC (#1:IBD096, 23-

year-old Japanese woman, UC-DAI mild; #2:IBD118, 65-year-old Japanese man, UC-DAI moderate) and from

healthy donors (#1:S-AKO07, 37-year-old Japanese man; #2: S-AKO17, 39-year-old Japanese woman) were

selected as representatives of each group (healthy, CD, and UC) on the basis of the principal coordinate analysis

of 16S rRNA sequencing data of saliva microbiota. Saliva samples were suspended in equal volume (w/v) of PBS

containing 20% glycerol/PBS, snap-frozen in liquid nitrogen, and stored at –80°C until use. The frozen stocks

were thawed, centrifuged at 3,300 g for 10 min at 4°C, suspended in PBS and orally inoculated into GF mice (100

µL per mouse). To isolate TH1-inducing bacterial strains, cecal contents from GF+CD#2 and GF+UC#2 mice

were serially diluted with PBS and seeded onto nonselective and selective agar plates (Schaedler, BHI, GAM,

CM0151, EG, BL, TS, and FS). After culture under anaerobic conditions (80% N2, 10% H2, 10% CO2) in an

anaerobic chamber (Coy Laboratory Products) at 37°C for 2 or 4 days, individual colonies were picked, 16S

rRNA gene region was amplified with the universal primers (27F: 5ʹ-AGRGTTTGATYMTGGCTCAG-3ʹ,

1492R: 5ʹ-GGYTACCTTGTTACGACTT-3ʹ) and sequenced. Individual isolates in the culture collection were

grouped into “strains” if their 16S rRNA gene sequences had 100% identity. The resulting strain sequences were

compared to those in the RDP database and to OTUs observed in fecal samples from GF+CD#2 and GF+UC#2

to determine closely related species or strains and corresponding OTUs. To prepare the bacterial mixture, bacterial

strains were individually grown in Schaedler (Kp-2H7, Ec-2B1), PYG (2D5, Ve-2E1, 2G7, 2E4) or EGF (Fu-21f,

2B11) broth to confluence and mixed at equal volumes of medium. The mixture of isolates was orally

administered to GF mice (approximately 1-2 × 108 CFU of total bacteria in 200 µL medium per mouse). All mice

receiving a given mixture of bacterial strains were maintained in a single gnotobiotic isolator. K. pneumoniae

3

BAA-2552, BAA-1705, 700721, 700603, and 13882 strains were purchased from American Type Culture

Collection (Manassas, VA, USA). K. pneumoniae KP-1 was isolated at the Scott A. Rice Laboratory (35).

KCTC2242 was obtained from the Korean Collection for Type Cultures (Daejeon, Korea). K. pneumoniae

34E1 was isolated from cecal contents of ampicillin-treated SPF mice at Kenya Honda’s laboratory. Ka-11E12

was initially identified as Enterobacter aerogenes based on 16S rRNA gene sequencing. However, because E.

aerogenes is phylogenetically very close to K. pneumoniae with >99% 16S rRNA gene sequence identity and

has recently been proposed to be reclassified as K. aeromobilis (27), we renamed the 11E12 strain K.

aeromobilis 11E12 (Ka-11E12) strain. K. pneumonia strains were grown at 37°C in Schaedler, Luria–Bertani

(LB) broth or on LB agar plates. For administration of heat-killed bacteria, Kp-2H7 cultured overnight was washed with autoclaved water, heat-killed at 105°C for 30 min and given to GF mice via the drinking water

(5x107 equivalent CFU/mL) for 3 weeks. Antimicrobial susceptibility testing was performed at SRL Inc.

(Tokyo, Japan) using the broth microdilution method according to the Clinical and Laboratory Standards

institute (CLSI) guidelines. The following antibiotics: ampicillin (Nacalai), tylosin (sigma), metronidazole

(Nacalai), vancomycin (Wako), spectinomycin (Nacalai), meropenem (Nacalai), clarithromycin (Tokyo

Chemical Industry), trimethoprim (Nacalai), streptomycin (Wako), gentamycin (Nacalai), polymyxin-B

(Nacalai), tetracycline (Nacalai) were further tested at wide range of concentrations (0.3-1000 µg/ml). Bacterial

suspension (1×105 CFU/mL in 100 µL) was inoculated into each well of 96-well plate containing serial three-

fold diluted antibiotics. After incubation at 37 oC for 24 h, The absorbance at 630 nm was measured using a

microplate reader (Bio Rad).

Intratracheal injection of Kp-2H7

SPF B6 mice were anesthetized with Isoflurane and placed in supine position. Under aseptic conditions, the

trachea was opened in midline by about 2 cms vertical incision and 10µL of either sterile PBS or Kp-2H7

suspension (1x106 CFU/10µL) was injected into the trachea with a sterile 30-gauge needle. Mice were sacrificed

7 days after bacterial inoculation, the lungs were collected for isolation of lymphocytes and histological

examination.

Isolation of lymphocytes and flow cytometry

Small and large intestines, lung and palate were collected. Intestines were opened longitudinally, washed with

PBS to remove all luminal contents. All the samples were incubated in 20 mL of Hanks’ balanced salt solution

(HBSS) containing 5 mM EDTA for 20 min at 37°C in a shaking water bath to remove epithelial cells. After

removal of remaining epithelial cells, muscular layers and fat tissues using forceps, the samples were cut into

small pieces and incubated in 10 mL of RPMI1640 containing 4% fetal bovine serum, 0.5 mg/mL collagenase D,

0.5 mg/mL dispase II, and 40 µg/mL DNase I (all from Roche Diagnostics) for 45 min at 37°C in a shaking water

bath. The digested tissues were washed with 10 mL of HBSS containing 5 mM EDTA, resuspended in 5 mL of

40% Percoll (GE Healthcare), and underlaid with 2.5 mL of 80% Percoll in a 15 mL Falcon tube. Percoll gradient

separation was performed by centrifugation at 850 g for 25 min at 25°C. Lymphocytes were collected from the

interface of the Percoll gradient and washed with RPMI1640 with 10% FBS and stimulated with 50 ng/mL PMA

4

and 750 ng/mL ionomycin (both from Sigma) in the presence of Golgistop (BD Biosciences) at 37°C for 4 h.

After dead cells were labeled with Ghost Dye 780 (Tonbo Biosciences), the cells were permeabilized and stained

with anti-CD3e (BV605; Biolegend), CD4 (BV510; Biolegend), CD8a (PE/Cy7; Bioledgend), TCRβ (BV421;

Biolegend), TCRgd (PE; Bioledgend), CD44 (BV785; Bioledgend), IFN-γ (FITC or PE/Cy7; Biolegend), IL-17A

(eFluor660; eBioscience), T-Bet (PE/Cy7; Bioledgend), RORγt (PE or APC; eBioscience) and Foxp3 (PerCP-

Cy5.5; eBioscience) using the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences) as

manufacturer's instructions. All data were collected on a BD LSRFortessa or FACSAria II (BD Biosciences) and

analyzed with Flowjo software (TreeStar). CD4+ T cells were defined as a CD4+ TCRβ+ CD3e+ subset within the

live lymphocyte gate.

Assessment of the antigen specificity of colonic LP TH1 cells The OmpX gene of K. pneumoniae was cloned by PCR, inserted into pET-DEST42, and expressed in the BL21

E. coli strain. His-tagged recombinant OmpX was purified using a nickel column. Percoll-enriched colonic LP

cells, which include T cells and antigen-presenting cells, isolated from GF or GF+Kp2H7 mice were ex vivo

stimulated with PMA and ionomycin, or stimulated with autoclaved in vitro cultured Kp-2H7, or recombinant

OmpX (5 µg/ml) in the presence of GolgiStop for 5 h and intracellularly stained for IFN-g and IL-17A as

described above.

Scanning electron microscopy

Intestines were washed with PBS, fixed in 2.5% glutaraldehyde buffered in 50 mM phosphate (pH 7.2), and

postfixed in 1% osmium tetroxide in 50 mM phosphate buffer (pH 7.2). Samples were dehydrated in an ethanol

series and substituted with isoamyl acetate. After dehydration, the samples were dried with a critical point dryer

(CPD 030; Leica Microsystems), coated with platinum, and observed under a scanning electron microscope (SU-

1510; Hitachi High-Technologies) at 5 or 10 kV. For all scanning electron microscopy studies, more than 10

areas per animal (3–5 animals from each group) were examined.

Colonization of antibiotic-treated mice

SPF mice (WT B6, Il10-/-, or Ifngr1-/-) were treated with or without ampicillin (200 mg/L), tylosin (500 mg/L),

metronidazole (500 mg/L), spectinomycin (200 mg/L) or vancomycin (200 mg/L) for 4 days prior to gavage

through the drinking water. Kp-2H7 or Ka-11E12 was grown to log phase in LB broth, and 1-2 x 108 CFUs were

used to inoculate mice. Feces were collected on day 1, 3, 7 14 and 21 post-gavage and fecal DNA was extracted

as above as part of 16S rRNA gene pyrosequencing. Confirmation of colonization was achieved by qPCR with

specific primers for each strain: Klebsiella (ompK36-3_F: 5ʹ-GCGACCAGACCTACATGCGT-3ʹ, ompK36-

3_R: 5ʹ-AGTCGAAAGAGCCCGCGTC-3ʹ); Kp-2H7 (sca4_298_F: 5ʹ-AGCACTAGCGGCTGTGGTAT-3ʹ,

sca4_298_R: 5ʹ-ACTTACTCGGGCCCTTGATT-3ʹ); Ka-11E12 (group_4037_F: 5ʹ-

5

CTTCGCCTTCATCAGCTTCA-3ʹ, group_4037_R: 5ʹ-TCATCATTAACGCGGGTCAG-3ʹ). At the end of

the experiment, colon tissue was collected and examined for TH1 cell frequency.

Preparation of colonic ECs and DCs Colon tissues were harvested, cut open longitudinally, and washed well with ice-cold PBS. Colonic epithelial

cells (ECs) were scraped using a glass slide, immediately frozen in liquid nitrogen and stored at −80°C until

further analysis. The residual tissues were incubated with 5 mM EDTA in HBSS at 37°C for 20 min with shaking

to completely remove ECs, cut into small pieces and incubated with RPMI1640 containing 4% fetal bovine serum,

0.5 mg/mL collagenase D, 0.5 mg/mL dispase II, and 40 µg/mL DNase I for 45 min at 37°C in a shaking water

bath. CD11c-positive cells were stained with anti-CD11c (APC; Biolegend) and enriched by MACS using anti-

APC beads (Miltenyi Biotec). Positively selected cells were further sorted on FACSAriaII, with a resulting

purity of around 97%. For the measurement of IL-18 production, EC fraction was dissociated from the colon

tissues treated with 5 mM EDTA for 20 min at 37°C on a shaker. Collected cells were washed with RPMI1640

containing 4% FBS, resuspended in 5 ml of 20% Percoll and overlaid on 2.5 ml of 40% Percoll in a 15-ml

Falcon tube. Percoll gradient separation was performed by centrifugation at 850 g for 25 min at 25°C. The

interface cells were collected and used as colonic ECs. Colonic ECs were suspended in RPMI 1640 containing

10% FBS and cultured in 24 well plates at 6 × 105 cells for 24 hours. Culture supernatants were collected and

the level of IL-18 was measured by ELISA (eBioscience).

RNA-seq and qPCR analysis

Total RNA was isolated from colonic ECs and DCs using TRIzol reagent (Invitrogen) as manufacturer's

instructions. For real-time qPCR analysis, cDNA was synthesized using ReverTra Ace qPCR RT Master Mix

(TOYOBO), and qPCR was performed using Thunderbird SYBR qPCR Mix (TOYOBO) on a LightCycler 480

(Roche). The following primer pairs were used: Actb, 5ʹ-TATGCCAACACAGTGCTGTC-3ʹ and 5ʹ-

ACCGATCCACACAGAGTACTTG-3ʹ; Gbp2, 5ʹ-TGCTGGATCTTTGCTTTGGC-3ʹ and 5ʹ-

AGTTAGCTCCGTCACATAGTGC-3ʹ; Gbp6, 5ʹ-AATGCCTTGAAGCTGATCCC-3ʹ and 5ʹ-

GTTCTTTGTCATGCGTTGGC-3ʹ; Ifi47, 5ʹ-GGCTCATTGCTTCAGACTTTCC-3ʹ and 5ʹ-

ACTGATCCATGGCAGTTACCAG-3ʹ; Cxcl9, 5ʹ-ATCATCTTCCTGGAGCAGTGTG-3ʹ and 5ʹ-

TTGTTGCAATTGGGGCTTGG-3ʹ; H2-Ab1, 5ʹ-TTGGCCTTTTCATCCGTCAC-3ʹ and 5ʹ-

ATTCGGAGCAGAGACATTCAGG-3ʹ; H2-DMb1, 5ʹ-CCCATCCAGACAGTGAAGGT-3ʹ and 5ʹ-

GCTGGAGGAATGAGACTTGC-3ʹ; Ifi208, 5ʹ-AGAACTTGCAGCTCGTGTTG-3ʹ and 5ʹ-

TGGTTCTACTTCCCAAGCTTCC-3ʹ; Ifng, and 5ʹ-

TGAGCTCATTGAATGCTTGG-3ʹ; Duox2,

5ʹ-GCGTCATTGAATCACACCTG-3ʹ

5ʹ-TGCGCCTGTTACTGTGATTG-3ʹ and 5ʹ-

AATGGAAAGCAGCAGACAGC-3ʹ; Tnfa, 5ʹ-TCATACCAGGAGAAAGTCAACCTC-3ʹ and 5ʹ-

GTATATGGGCTCATACCAGGGTTT-3ʹ. For RNA-seq, RNA library preparation was performed using a

NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s

instructions. After assessing the library quality, sequencing was carried out on a HiSeq 1500 system (Illumina)

6

using single-ended 50-bp reads. The sequenced reads were mapped to the mouse reference genome (mm9, NCBI

build 37) and normalized to fragments per kilobase per million reads (FPKM) values using the Tophat and

Cufflinks software pipeline. The heatmap in Fig. 2F shows the relative abundance (Z-score) of genes that were

upregulated (>2-fold, FPKM value ≥ 0.1) in Kp-2H7-monocolonized mice versus GF and BAA2552-

monocolonized mice. The heatmap in fig. S6A shows the relative abundance (Z-score) of genes that were

commonly upregulated (>2-fold, FPKM value ≥ 0.1) in Kp-2H7-monocolonized WT mice versus Kp-2H7-

monocolonized Myd88-/- mice. The upregulated genes (57 genes in Fig. 2G left, 47 genes in Fig. 2G right or 55

genes in fig. S6B) were subjected to GO enrichment analysis using the DAVID Bioinformatics Resources 6.8

(36).

For stimulation of colonic ECs with cecal suspensions, frozen cecal contents from GF or GF+Kp-2H7

mice were thawed and well suspended in 4 times volume (w/v) of sterile water. After centrifugation (5,000 g

for 10 min), supernatants were passed through a 0.22 µm filter and used as bacteria-free cecal suspensions. The

CMT93 mouse colonic epithelial cell line was obtained from ATCC, and cultured at 2 x 105 cells in 300 µl

RPMI containing 10% FBS in 24 well plates with 10 µl cecal suspensions. Total RNA was isolated at 0, 1, 3, 6,

and 12 h after the addition of cecal suspensions using TRIzol reagent (Invitrogen) as manufacturer’s

instructions, subjected to real-time qPCR analysis.

Histological analysis

For fluorescence in situ hybridization staining (FISH) of Kp-2H7 and Ka-11E12, colon tissue was fixed with

methanol-Carnoy’s solution and embedded in paraffin. The sections were treated with 0.1 M HCl and hybridized

with the 5ʹ Alexa 488-labeled EUB338 (5ʹ-GCTGCCTCCCGTAGGAGT-3ʹ) probe and stained with rhodamine

labeled Ulex Europaeus Agglutinin I (UEA1; Vector Laboratories). All the sections were counterstained with

DAPI, mounted with Fluoromount/Plus (Diagnostic BioSystems), and visualized using a Leica TCS SP5 confocal

microscope. To evaluate development and severity of colitis and pneumonia, mice were sacrificed at 5 weeks

after oral inoculation or 7 days after intratracheal injection. Colons and lung were fixed with 4%

paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The degree of colitis

was graded according to the following criteria: inflammatory cell infiltration (score, 0–4), mucosa thickening

(score, 0–4), goblet cell depletion (score, 0–4), crypt abscess (score, 0–4) and destruction of architecture (score,

0–4). The final histological score was defined as the sum of the scores of these parameters. The pneumonia score

was determined as the sum of the scores of two sections, alveoli (no change 0, edema 1, Inflammatory cells in

alveolar lumina 2, Inflammatory destruction of alveoli with lung abscess 3) and bronchioles (no change 0, mild

inflammation in the wall 1, severe inflammation in the wall with luminal slough 2, severe inflammation with

luminal slough and peribronchial inflammation 3).

Bacterial genome sequencing

The genome of Klebsiella strains was extracted by a similar method described above as part of 16S rRNA gene

pyrosequencing. The genome sequences were determined by the whole-genome shotgun strategy using PacBio

7

RSII and Illumina MiSeq sequencers. The genomic DNA was sheared to obtain DNA fragments. Template DNA

was prepared according to each supplier’s protocol. Obtained RSII reads were subjected to de novo-assembly

using HGAP3. MiSeq reads (2 x 300 nt) were mapped onto the RSII assembled contigs to correct low quality

regions. To evaluate the phylogeny of isolated strains, we downloaded 54 complete genomes and 15 draft

genomes from NCBI, which included 59 K. pneumoniae strains, 3 K. variicola strains, 1 K. michiganensis strains,

3 K. oxytoca strains, 1 K. quasipneumoniae strains and 2 K. aeromobilis (E. aerogenes) strains. Phylogenetic

trees were constructed based on the Mash distance (37) using neighbor-joining method.

MLST, wzi and wzc sequence typing

The genome sequences of each strains are aligned against MLST database

(http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Sequence-based capsular (K) typing was carried out based on

sequencing of wzi or wzc genes (38, 39).

Identification of orthologous groups correlating with TH1-inducing activity

The strains of Klebsiella spp. were categorized into strong, medium, and weak inducers for TH1 cells. To

select the enriched orthologs, we calculated a strong-inducer index for the k-th ortholog group, SIIk (Strong

Inducer Index), to facilitate the identification of genes that potentially contribute to TH1 induction. SIIk was

defined as: SIIk=[f(s)k+f(m)k]/[f(m)k+f(w)k], in which f(s)k, f(m)k, and f(w)k represent the frequency of the

k-th ortholog groups present among strong, medium, and weak TH1 inducers, respectively. We selected the

orthologs with KII > 2; and for each ortholog, we performed a G-test (with FDR correction) in comparison with

randomized draws, and finally selected the ones with q<0.1.

Bacterial motility assay

Bacterial motility assay was performed using semi-solid LB medium containing 0.25% agar in 14 ml tube.

Bacteria cultured in LB broth medium was inoculated into a semi-solid LB medium by a straight wire, making a

single stab down the center of the tube to about half the depth of the medium. After incubation at 37 ℃ for 24

hours, bacteria which showed diffuse and hazy spread throughout the medium were determined as motile bacteria.

Bacterial flagella were detected by HEK-BlueTM mTLR5 cells (InvivoGen), following the manufacturer’s

protocol.

Statistical analysis

All statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.) or JMP

software v.12 (SAS Institute, Inc.) with two-tailed unpaired Student’s t-test (parametric), Wilcoxon rank-sum test

(non-parametric) and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (3 or more

groups, parametric).

IL-17

fig. S1

IFN-

γ

GF

D

A

B

0 102 103 104 105

0

102

103

104

105

1.14

0.01975.28

93.60 102 103 104 105

0

102

103

104

105

12.4

4.071.39

82.10 102 103 104 105

0

102

103

104

105

3.58

0.2375.22

910 102 103 104 105

0

102

103

104

105

15.1

0.05265.4

79.50 102 103 104 105

0

102

103

104

105

42.3

4.830.624

52.3

0 102 103 104 105

0

102

103

104

105

2.61

1.1133.8

62.50 102 103 104 105

0

102

103

104

105

17.5

33.22.14

47.20 102 103 104 105

0

102

103

104

105

5.04

1.6533.7

59.70 102 103 104 105

0

102

103

104

105

15.1

0.53534.8

49.60 102 103 104 105

0

102

103

104

105

34.6

31.63.75

30.1

T-bet RORγt Foxp3 CD44

GF

+Kp-

2H7

GF

+Kp-

2H70

10

20

30

40

Colon SI

% IF

N-γ+ C

D4 T

0

10

20

30

40

% IF

N-γ+ C

D4 T

GF

+Kp-

2H7

palate

EG

F+K

p-2H

7G

F+K

p-2H

70

10

20

30

40

IQI BALB/c

% IF

N-γ+ C

D4 T

F

SI_1

SI_2

SI_3

SI_4

Cecu

mCo

lon

Fece

s0

50

100

150

Kp-2

H7 D

NA c

onc.

�ѥ

g/g

cont

ent)

0 102 103 104 105

0

102

103

104

105

87.2

6.410.74

5.66

0 102 103 104 105

0

102

103

104

105

55

37.11.47

6.38

0 102 103 104 105

0

102

103

104

105

92.7

0.8580.863

5.540 102 103 104 105

0

102

103

104

105

2.02

1.261.56

95.20 102 103 104 105

0

102

103

104

105

18.6

2.334.67

74.4

0 102 103 104 105

0

102

103

104

105

6.7

3.5537.3

52.5

0 102 103 104 105

0

102

103

104

105

24.3

0.271.2

74.3

0 102 103 104 105

0

102

103

104

105

17.5

0.07844.42

78

TCRγδ

IFN-

γ

GF

GF+Kp-2H7

TCRβ CD8α

IL-1

7

TCRβ0 102 103 104 105

0

102

103

104

105

90.2

4.470.918

4.390 102 103 104 105

0

102

103

104

105

1.87

0.9774.5

92.7

IL-1

7

IFN-

γ

CD8α

gated on CD3ε+ cells gated on CD3ε+ TCRβ+ cells

C

gated on CD3ε+ TCRβ+ CD4+ cells

****

ns

GF+Kp-2H7

B6 B6 Colon

ns

fig. S1. Characteristics of colonic TH1 cells induced by Kp-2H7. (A) Representative flow cytometry dot plots showing expression of IFN-γ, IL-17A, T cell receptor β (TCRβ), TCRγδ, and CD8α by colonic LP CD3ε+ T cells or CD3ε+7&5ћ+ T cells from GF mice and GF+Kp-2H7 mice. (B) Expression of IFN-γ, IL-17,7�EHW��525ќW��)R[S���DQG�&'���E\�FRORQLF�&'�ε+ TCRβ+ CD4+ T cells from GF mice and GF+Kp-2H7mice. (C) Bacterial DNAs were extracted from fecal pellets and luminal contents from the small intestine (SI, subdivided into 4 segments from proximal to distal), cecum, and colon of GF+Kp-2H7 mice (n=5). Kp-2H7 DNA concentration was determined by q-PCR. (D-F) The percentage of IFN-γ+ cells within CD4+ T cells (TH1 cells) in the colonic LP (D and F), SI LP (D), and palate (E) from B6 mice (D and E), IQI mice (F), and BALB/c mice (F) monocolonized with Kp-2H7. Symbols represent individual mice. Error bars indicate mean ± SD. *P < 0.05; ***P < 0.001; ns, not significant (P > 0.05), one-way ANOVA with post hoc Turkey's test. Data represent at least 2 independent experiments with similar results.

fig. S2

0.1 1 10 100 10000.0

0.5

1.0

Abx conc. (μg/ml)

Abso

rban

ce 6

30nm

TylMNZ

CAM

VCMSpc

TMPSMGMPL-BTC

MEPM

Amp

0

APenicillins

AmpicillinPiperacillin

CephemsCefaclorCefpodoxime-ProxetilCefazolinCefotiamCefotaximeCeftazidimeCefpiromeCefmetazoleFlomoxef

CarbapenemsImipenem /CilastatinMeropenem

MonobactamsAztreonam

β-lactamase inhibitorsAmoxicillin /ClavulanateSulbactam /Cefoperazone

AminoglycosidesGentamicinAmikacin

TetracyclinsMinocycline

OthersSulfamethoxazole-TrimethoprimLevofloxacinFosfomycin

>16≤ 8

≤ 4≤ 11

≤ 0.5≤ 0.5≤ 1≤ 4≤ 8≤ 8

≤ 0.25≤ 0.25

≤ 1

≤ 4

≤ 4

≤ 2≤ 8

≤ 2

≤ 20≤ 116

RS

SSSSSSSSS

SS

S

S

S

SS

S

SSI

MIC (μg/ml) CLSIAntibioticB

fig. S2. Kp-2H7 is resistant to multiple antibiotics. (A) Kp-2H7 was incubated at 37°C in a 96-well plate inthe presence of different concentrations of antibiotics for 24 h. Bacterial growth was determined by measuringthe absorbance at 630 nm. Data represent the mean ± the SD. Data represent at least 3 independent experi-ments. Amp, ampicillin; Tyl, tylosin; MNZ, metronidazole; VCM, vancomycin; Spc, spectinomycin; MEPM, meropenem; CAM, clarithromycin; TMP, trimethoprim; SM, streptomycin; GM, gentamycin; PL-B, polymyx-in-B; TC, tetracycline. (B) Antibiotic sensitivity of Kp-2H7.

fig. S3

fig. S3. Kp-2H7 has colitogenic potential. (A, B) SPF B6 WT or Il10-/- mice were continuously treated with or without ampicillin (200 mg/L) in drinking water, starting 4 days before oral administration of 2 × 108 CFU Kp-2H7. Three weeks after Kp-2H7 adminis-tration, colon tissues were harvested and examined by hematoxylin and eosin (H&E) staining. Representative H&E staining of the proximal colon (A) and histological colitis scores (B) are shown. (C-E) GF Il10-/- mice were colonized with Kp-2H7, Ec-2B1, or 6-mix. One week after colonization, percentages of IFN-γ+ CD4 T (TH1) cells and IFN-γ+ IL-17+ CD4 T cells in the colonic LP (C), and relative expression of Tnfa mRNA in the colonic ECs (D) were examined by FACS and qPCR, respectively. (E) Three weeks after Kp-2H7 administration, colon tissues were harvested and examined by hematoxylin and eosin (H&E) staining. Scale bars, 200 μm (A and E). Symbols represent individual mice. Error bars indicate mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05), one-way ANOVA with post hoc Turkey’s test. Data representat least 2 independent experiments with similar results.

SPF Il10-/- +Amp+Kp-2H7SPF Il10-/- +Amp

SPF WT +Amp+Kp-2H7SPF WT +AmpA

Hist

olog

ical s

core

SPF+Amp

2

8

6

4

10

0

Kp-2

H7

Kp-2

H7Co

nt

Cont

Il10-/-WT

***

ns

B

C

GF Il10-/- +Kp-2H7GF Il10-/- GF Il10-/- +Ec-2B1E

** **

0

5

10

15

20

0

1

2

3

Kp-2

H7Ec

-2B1

GF

Il10 -/-

6-m

ix

IFN-

γ+ CD4

T

IFN-

γ+ IL

-17+ C

D4 T*** *****

***

0

0.05

0.10

0.15

0.20

Rela

tive

expr

essio

n of

Tnf

a ****

***D

GF Il10-/- +6-mix

Kp-2

H7G

F

WT

Kp-2

H7Ec

-2B1

GF

Il10 -/-

6-m

ix

Kp-2

H7G

F

WTKp

-2H7

Ec-2

B1

GF

Il10 -/-

6-m

ix

Kp-2

H7G

F

WT

ns*** **

fig. S4

IL-17

IFN-

γSPF SPF+Kp-2H7

%IF

N-γ

+ CD

4 T

0

10

20

30

40

GF

+Kp-

2H7

Colon

SPF

+Kp-

2H70

10

20

30

40Lung

%IL

-17+ C

D4

T

02468

1012

GF

+Kp-

2H7

02468

1012

SPF

+Kp-

2H7

0 102 103 104 105

0

102

103

104

105

0.862

0.03146.5

92.60 102 103 104 105

0

102

103

104

105

9.9

0.81411.4

77.8

0 102 103 104 105

0

102

103

104

105

1.16

0.02915.55

93.30 102 103 104 105

0

102

103

104

105

2.75

1.5333.4

62.3

IL-17

IFN-

γ

%IF

N-γ

+ CD

4 T

%IL

-17+ C

D4

T

GF GF+Kp-2H7

SPF+PBS (Lung) SPF+Kp-2H7 (Lung)

TH1A TH17

TH1 TH17

B C

+PBS

+Kp-

2H70

2

4

6

Hist

olog

ical

scor

e

*

**

***

***

*

fig. S4. Pneumonia induced by Kp-2H7. Kp-2H7 (1 × 106 CFU) was intratracheally injected into the lungs of SPF WT B6 mice. 7 days after injection, lung tissues were harvested and examined for TH1 and TH17 responses and for lung pathology. (A) Frequencies of IFN-γ+ and IL-17+ cells among lung CD4+

TCRβ+ T cells from mice inoculated with Kp-2H7 are shown. To compare the T-cell response to Kp-2H7 in the colon, frequencies of IFN-γ+ and IL-17+ cells among colon CD4+ TCRβ+ T cells from mice monocol-onized with Kp-2H7 are shown. (B, C) H&E staining (B) and histological scores (C) of the lung of PBS- or Kp-2H7-injected WT SPF mice. Scale bar, 200 μm. Symbols represent individual mice. Error bars indicate mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student's t-test. Data represent at least 2 independent experiments with similar results.

fig. S5

7 14 21

108

109

1010

1011

CFU/

g fe

ces

Kp-2H7Ec-2B1BAA-2252

KP-1KCTC2242

0days after inoculation

B

fig. S5. Phylogenetic relationship among K. pneumoniae strains used in this study. (Α) The whole-genome phylogenetic analysis of K. pneumoniae strains was performed based on mashdistance. Phylogenetic tree was constructed by neighbor-joining method. Klebsiella strains categorized into strong, medium, and weak inducers for TH1 cells are marked in red, yellow, and blue, respectively. (Β) The indicated K. pneumoniae strains were gavaged into GF mice, and the bacterial load of feces wasdetermined over time by enumeration of colony forming units (CFU).

A

K.pneu

moniae

KPNIH27

K.pneu

moniae

DHQP1002

001

K.pneu

moniae

strai

n PB15

7

K.pneu

moniae

47AV

RK.

pneu

moniae

IS53

K.pn

eum

oniae

436

KPN

E 40

5 21

4 29

23

K.pn

eum

onia

e st

rain

CCB

H133

27

K.pneumoniae strain KPO

I-1 3 124243G

K.pneumoniae J1

K.pneumoniae Kp52.145

K.pneumoniae MGH 18

K.pneumoniae UCLAOXA232KP

K.pneumoniae KPNIH29

K.pneumoniae KPNIH39

K.pneumoniae 68BO

K.pneumoniae KpN01K.pneumoniae SKGH01K.pneumoniae Kpn223

K.pneumoniae MS6671K.pneumoniae AATZP

K.pneumoniae HK787

K.pneumoniae Kp13K.pneumoniae KPNIH31

K.pneumoniae HS11286

K.pneumoniae JM45

K.pneumoniae ATCC BAA-2146

K.pneu

moniae

UHKPC33

K.pneumoniae CAV1596

K.pneumoniae KPNIH33

K.pneu

moniae

UHKPC07

K.pn

eum

onia

e M

GH

43

K.pn

eum

oniae

CAV

1344

K.pn

eumo

niae b

laNDM

-1 K.pneumoniae UCI70

K.pneumoniae 65BO

K.pn

eum

onia

e Kp

n555

K.pn

eum

onia

e st

rain

k17

65K.

pneu

mon

iae

stra

in k

1954

K.pneumoniae PM

K1

K.pneumoniae KP617

K.pneumoniae NUHL24835

K.pneumoniae BR

K.pneumoniae KP36

K.pneumoniae ED2

K.pneumoniae RJF999

K.pneumoniae ATCC43816 KPPR1

K.pneumoniae RJF293

K.pneumoniae 55BGK.pneumoniae 234-12

K.pneumoniae TGH8

K.pneumoniae W14

1388

2Kp

-2H7

BAA-1705

KP-

1

Kp-40B3

KCTC 2242

34E1

700721

K.pneumoniae strain k1982

K.pneumoniae subsp. pneum

oniae 1158

K.pn

eumon

iae 34

618

TH1 induction

WeakMediumStrong

E.aerogenes EA1509EE.aerogenes KCTC 2190

K.oxytoca CAV1374K.oxytoca CAV1015

K.michiganensis KCTC1686

K.oxytoca JKo3

K.variicola DX120E

K.pneumoniae YH43

K.variicola HKUOPLA

700603

BAA-2552K.variicola GJ1

Ka-11E12

fig. S6

fig. S6. Klebsiella antigen-specific TH1 cell induction and epithelial IL-18 production by Kp-2H7. (A) Colonic LP cells, which include T cells and antigen-presenting cells (APCs), were isolated from GF+Kp2H7 mice and stimulated ex vivo with GolgiStop (GS) only, with PMA and ionomycin (P/I), with auto-claved in vitro cultured Kp-2H7 (Kp-2H7 lysate), or with recombinant OmpX protein, which was reported to be one of the dominant Klebsiella antigens (Chen et al., The Journal of Immunology, Vol. 192 (1 Supplement) 141.12, 2014). IFN-γ and IL-17A expression was analyzed by FACS as a readout for T cell receptor (TCR) activation. Although less efficient than P/I, the Kp-2H7 lysate and recombinant OmpX evoked a IFN-γ response in a significant number of cells. (B) Colonic epithelial cells were collected from WT GF mice monocolonized with Kp-2H7 for 3 weeks, and cultured ex vivo for 24h. Culture supernatants were examined for IL-18 production by ELISA. Symbols repre-sent individual mice. Error bars indicate mean ± SD. *P < 0.05; ***P < 0.001, one-way ANOVA with post hoc Turkey’s test (A) and Student’s t-test (B).

A

0 102 103 104 105

0

102

103

104

105 0.678 0.0549

4.7894.50 102 103 104 105

0

102

103

104

105 0 0

0.03211000 102 103 104 105

0

102

103

104

105 0.0142 0

0.070899.90 102 103 104 105

0

102

103

104

105 0 0

0.0145100

0 102 103 104 105

0

102

103

104

105 2.4 1.4

33.762.50 102 103 104 105

0

102

103

104

105 0.0445 6.35e-3

0.10899.80 102 103 104 105

0

102

103

104

105 0.344 0.167

3.795.80 102 103 104 105

0

102

103

104

105 0.0658 0.0292

1.3798.5

IFN-γ

IL-1

7

P/Ι GS only Kp-2H7 lysate OmpX protein

GF

GF+Kp-2H7

GS

only

Kp-2

H7O

mpX

% IF

N-γ+ C

D4 T

0

2

4

6

GS

only

Kp-2

H7O

mpX

GF GF+Kp-2H7

****

B

0

100

200

300

IL-1

8 (p

g/m

l)

GF GF+Kp-2H7

*

colonic ECs

fig. S7

fig. S7. Gene expression profile of colonic DCs and ECs in GF+Kp-2H7 mice in comparison to GF+BAA-2552 mice. Differential gene expression in the colonic ECs and DCs from WT B6 mice mono-colonized with Kp-2H7 or BAA-2552 for 1 week was analyzed by RNA-seq. Gene ontology (GO) terms significantly enriched in up-regulated gene sets in the colonic ECs and DCs from GF+Kp-2H7 mice in comparison to GF and GF+BAA-2552 mice are shown.

0 4 6 8 102-log10 (p value)

Biological processCellular componentMolecular function

Heme bindingGTP binding

Oxygen bindingGTPase activityGolgi apparatus

Blood microparticleMHC class II protein complex

Response to virusInnate immune response

Cellular response to interferon-gammaAntigen processing and presentation

Negative regulation of T cell proliferationImmune response

Defense response to protozoanDefense response to Gram-positive bacterium

Antigen processing and presentation ofexogenous peptide antigen via MHC class II

Adaptive immune responseDefense response to virus

Defense responseCellular response to interferon-beta

Immune system process

colonic ECs

colonic DCs

0 5 10-log10 (p value)

GTP binding

Cytokine activityHydrolase activity

GTPase activityExtracellular region

Cell surfaceMembrane

Symbiont-containing vacuole membraneDefense response to virus

Defense responseImmune response

Defense response to Gram-positive bacteriumResponse to virus

Adhesion of symbiont to hostCellular response to lipopolysaccharideCellular response to interferon-gamma

Cellular response to interferon-betaDefense response to protozoan

Biological processCellular componentMolecular function

fig. S8

fig. S8. Gene expression profile of colonic ECs in Kp-2H7–colonized mice in comparison to

innate Myd88- or Tlr4-deficient mice.

(Α, Β) Colonic ECs were collected from WT, Myd88-/-, Tlr4-/- and Myd88-/-Trif-/- mice monocolonized withor without Kp-2H7 for 3 weeks, and gene expression profiles were analyzed by RNA-seq. (Α) Heatmapcolors represent the z-score normalized FPKM values for each gene (red and blue indicate high and low expression, respectively). (Β) Gene ontology (GO) terms significantly enriched in up-regulated gene setsin cells from WT+Kp-2H7 mice are shown.

Reg3bReg3gIdo1Wfdc18Gbp2Duoxa2Ifi47Pisd-ps1Hspa12aIgtpCd74Zbp1H2-AaGbp6H2-DMb12210407C18RikH2-Ab1Gbp7Duox2Gbp8H2-Eb1Psmb8GzmaH2-DMaGm5431Duox1Apol9aOas3Herc6Apol9b

colonic ECs W

T-G

FW

T+K

p-2H

7

Myd88

-/-

+Kp2

H7

Tlr4

-/-

+Kp2

H7

Myd88

-/- Trif

-/-

+Kp2

H70

5

10

15

Hem

e bi

ndin

gCa

rboh

ydra

te b

indi

ngPe

ptid

e an

tigen

bin

ding

MHC

cla

ss II

pro

tein

com

plex

bin

ding

GTP

bin

ding

GTP

ase

activ

ityLy

soso

me

Exte

rnal

sid

e of

pla

sma

mem

bran

eEn

dopl

asm

ic re

ticul

um m

embr

ane

Late

end

osom

e m

embr

ane

Extra

cellu

lar r

egio

nM

ultiv

esicu

lar b

ody

Sym

bion

t-con

tain

ing

vacu

ole

mem

bran

eM

HC c

lass

II p

rote

in c

ompl

exLi

pid

trans

port

Inna

te im

mun

e re

spon

seIn

flam

mat

ory

resp

onse

Prot

eolys

is in

volve

d in

cel

lula

r pro

tein

cat

abol

ic pr

oces

sDe

fens

e re

spon

se to

viru

sNe

gativ

e re

gula

tion

of T

cel

l pro

lifera

tion

Acut

e-ph

ase

resp

onse

Posit

ive re

gula

tion

of T

cel

l diff

eren

tiatio

nCh

aper

one

med

iate

d pr

otei

n fo

ldin

g re

quiri

ng c

ofac

tor

Adhe

sion

of s

ymbi

ont t

o ho

stDe

fens

e re

spon

se to

Gra

m-p

ositiv

e ba

cter

ium

Defe

nse

resp

onse

to p

roto

zoan

Resp

onse

to v

irus

Resp

onse

to in

terfe

ron-

gam

ma

Cellu

lar r

espo

nse

to in

terfe

ron-

gam

ma

Imm

une

resp

onse

Defe

nse

resp

onse

Antig

en p

roce

ssin

g an

d pr

esen

tatio

n of

pep

tide

or p

olys

acch

arid

e an

tigen

via

MHC

cla

ss II

Antig

en p

roce

ssin

g an

d pr

esen

tatio

n of

exo

geno

us p

eptid

e an

tigen

via

MHC

cla

ss II

Antig

en p

roce

ssin

g an

d pr

esen

tatio

nCe

llula

r res

pons

e to

inte

rfero

n-be

taIm

mun

e sy

stem

pro

cess

Biological processCellular componentMolecular function

AB

-log1

0 (p

val

ue)

Z-score

2-2 0

fig. S9

GF

+Kp-

2H7

+BAA

-255

2

Rela

tive

expr

essio

nRe

lativ

e ex

pres

sion

Rela

tive

expr

essio

nRe

lativ

e ex

pres

sion

Rela

tive

expr

essio

nRe

lativ

e ex

pres

sion

colonic DCs(1 week)

colonic ECs(1 week)

WT-

GF

WT+

Kp-2

H7Myd88

-/-+K

p-2H

7Tlr4

-/-+K

p-2H

7Myd88

-/- Trif

-/-

+Kp-

2H7G

F+K

p-2H

7+B

AA-2

552

colonic ECs(3 weeks)

A B

Rela

tive

expr

essio

n

H2-Ab1

Cxcl9

Gbp6

Rela

tive

expr

essio

nRe

lativ

e ex

pres

sion

Rela

tive

expr

essio

n

Ifi47

days after inoculation0 2173

colonic ECs C

fig. S9. Gene expression of colonic ECs and DCs and kinetics of TH1 cell induction in Kp-2H7 colonized mice. (A, B) Relative expression of selected genes normalized to Actb in colonic ECs and DCs from the indicated mice at the indicated time points after bacterial inoculation was assessed by qPCR. (C) The kinetics of the accumulation of TH1 cells in GF+Kp-2H7 mice was examined by flow cytometry. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA with post hoc Turkey’s test (A) and Student’s t-test vs GF mice (B and C).

0

2

4

6

8 Gbp2

0

5

10

15 Cxcl9

0

2

4

6

8 Gbp6

0

2

4

6

8 Ifi47

012345

H2-Ab1

Duox2

0

5

10

15 Gbp2

0

2

4

6

8 Cxcl9

0

1

2

3

4H2-DMb1

012345 Gbp6

0

1

2

3 Ifi47

0

1

2

3H2-Ab1

0

2

4

6 Gbp2

0

1

2

3

4 Cxcl9

00.51.01.52.02.5 Gbp6

0

1

2

3

4Ifng

0

1

2

3

4 Ifi47

0

1

2

3Ifi208

**

*

*

**

*

**

****

*

**

**

***

*****

***

0

1

2

3

4 ***

0

5

10

15

0

5

10

15

05

10152025

05

10152025

14

Duox2

0

2

4

6

8

Rela

tive

expr

essio

n%

IFN-

γ+ in

CD4

T TH1

0

10

20

30

40

days after inoculation0 2173

colonic LPLs

14

***

ns*

***

**

*

*

**

***

***

***

***

*

*

**

***

*

**

***

***

****

***

fig. S10

fig. S10. Upregulation of IFI genes in a colonic epithelial cell line by bacteria-free cecal suspen-sions from GF+Kp-2H7 mice in vitro. Cecal contents were collected from GF+Kp-2H7 and GF mice, suspended in water, and filtered through a 0.22 μm filter to exclude bacterial cells. The CMT93 colonicepithelial cell line was stimulated with the filtered bacterial cell-free cecal suspensions and expression ofIFN-inducible genes was examined by qPCR. Cecal suspensions from GF+Kp2H7 mice induced signifi-cant upregulation of IFN-inducible genes, whereas those from GF mice had negligible effects, suggest-ing that Kp-2H7 produces extracellular innate immune ligands that can activate intestinal ECs.

0 1 3 6 12

Gbp2

hours after stimulation

Ifi47Gbp6

0 1 3 6 12 0 1 3 6 120

5

10

15

Rel

ativ

e ex

pres

sion

0

1

2

3 GF CecalKp-2H7 Cecal

2

4

6

8

0

fig. S11

0.1 1 10 100 10000.0

0.5

1.0

Abx conc. (μg/ml)

OD6

30

TylosinMNZ

CAM

VCMSpec

TMPSMGMPL-BTC

MEPM

Amp

0

A

C GF WT

GF Il10-/- +Ka-11E12

GF WT +Ka-11E12

GF Il10-/-

Hist

olog

ical s

core

15

10

4

20

0

Ka-1

1E12

Ka-1

1E12

Cont

Cont

Il10-/-WT

***

ns

PenicillinsAmpicillinPiperacillin

CephemsCefaclorCefpodoxime-ProxetilCefazolinCefotiamCefotaximeCeftazidimeCefpiromeCefmetazoleFlomoxef

CarbapenemsImipenem /CilastatinMeropenem

MonobactamsAztreonam

β-lactamase inhibitorsAmoxicillin /ClavulanateSulbactam /Cefoperazone

AminoglycosidesGentamicinAmikacin

TetracyclinsMinocycline

OthersSulfamethoxazole-TrimethoprimLevofloxacinFosfomycin

>16≤ 8

8≤ 1> 4

≤ 0.5≤ 0.5≤ 1≤ 416≤ 8

≤ 0.25≤ 0.25

≤ 1

>16

≤ 4

≤ 2≤ 8

≤ 2

≤ 20≤ 116

RS

SSRSSSSSS

SS

S

R

S

SS

S

SSI

MIC (μg/ml) CLSIAntibioticB

fig. S11. Ka-11E12 is resistant to multiple antibiotics and has colitogenic potential. (A) Ka-11E12 was incubated at 37°C in a 96-well plate in the presence of different concentrations of antibiotics for 24 h. Bacterial growth was determined by measuring the absorbance at 630 nm. Data represent the mean ± the SD. Data represent at least 3 independent experiments. Amp, ampicillin; Tyl, tylosin; MNZ, metronidazole; VCM, vancomycin; Spc, spectinomycin; MEPM, meropenem; CAM, clari-thromycin; TMP, trimethoprim; SM, streptomycin; GM, gentamycin; PL-B, polymyxin-B; TC, tetracycline. (B) Antibiotic sensitivity of Ka-11E12. (C, D) GF WT or Il10-/- mice were monocolonized with Ka-11E12. Five weeks after Kp-2H7 administra-tion, colon tissues were harvested and examined by H&E staining. Representative H&E staining of the proximal colon (C) and histological colitis scores (D) are shown. Symbols represent individual mice. Error bars indicate mean ± SD. ***P < 0.001; ns, not significant (P > 0.05), one-way ANOVA with post hoc Turkey’s test. Scale bars, 200 μm.

D

fig. S12

A

B

med

ium

0

50

100

150

med

ium

Ka-1

1E12

Kp-2

H722

52KC

TC22

42

Rela

tive

TLR5

-st

imul

atin

g ac

tivity ***

nsnsns

Kp-2

H7

BAA-

2552

KCTC

2242

Ka-1

1E12

Ec-2

B1

fig. S12. Motile characteristics of Ka-11E12. In vitro-cultured Ka-11E12 bacteria were subjected to transmission electron microscopy (A) and a motility assay (B). (C) HEK-Blue TLR5 cells were incubated with culture supernatant of indicated strains and activation of TLR5 was evaluated by a luminescence assay. Error bars indicate mean ± SD. ***P < 0.001; ns, not significant (P > 0.05), one-way ANOVA with post hoc Turkey's test. Scale bar, 1μm.

C

Ka-11E12

Fig. 2D

TH1 induction

WeakMediumStrong

Fru

ctos

e,

gala

ctito

l-rel

ated

Man

nose

-re

late

d

Typ

e V

Ise

cret

ion

syst

em

Long

-cha

infa

tty a

cid

7007

21B

AA

-170

5

Kp-

2H7

7006

03

KP

-1K

CT

C 2

242

Kp-

40B

3

BA

A-2

552

Ka-

11E

12

1338

234

E1

PAN_4600PAN_2636PAN_3577PAN_5131PAN_4950PAN_709PAN_2321PAN_2474PAN_2584PAN_5365PAN_5089PAN_4304PAN_719PAN_3769PAN_4968PAN_5317PAN_4139PAN_4841PAN_617PAN_2232PAN_1642PAN_1403PAN_1756PAN_1987PAN_1932PAN_5164PAN_2549PAN_3824PAN_1946PAN_5526PAN_5285PAN_4439PAN_2220PAN_194PAN_1374PAN_1370PAN_5334PAN_3404PAN_5448PAN_4787PAN_3524PAN_3662PAN_4857PAN_575PAN_5040PAN_5163PAN_4658PAN_5054PAN_5137PAN_4653PAN_2405PAN_5372PAN_4704PAN_3965PAN_4789PAN_3737PAN_454PAN_5072PAN_4872PAN_4471PAN_3091

TH1 induction

WeakMediumStrong

Fru

ctos

e,

gala

ctito

l-rel

ated

Man

nose

-re

late

d

Typ

e V

Ise

cret

ion

syst

em

Long

-cha

infa

tty a

cid

7007

21B

AA

-170

5

Kp-

2H7

7006

03

KP

-1K

CT

C 2

242

Kp-

40B

3

BA

A-2

552

Ka-

11E

12

1338

234

E1

PRISM

CD UC Non-IBD

RPKM

0

20

40

60

80

100

PAN_4950PAN_2405PAN_5054PAN_4653PAN_4787PAN_3965PAN_1987PAN_4872PAN_5163PAN_3662PAN_5448PAN_4704PAN_5137PAN_3737PAN_2321PAN_4471PAN_454PAN_5372PAN_2232PAN_1403PAN_4857PAN_617PAN_5526PAN_194PAN_4439PAN_1374PAN_2220PAN_3091PAN_5072PAN_1932PAN_5164PAN_5285PAN_4789PAN_3524PAN_4968PAN_5040PAN_575PAN_4139PAN_1946PAN_2549PAN_3824PAN_4841PAN_5317PAN_3769PAN_1370PAN_4304PAN_719PAN_2584PAN_5089PAN_4658PAN_2474PAN_1642PAN_5365PAN_3404PAN_5131PAN_4600PAN_709PAN_3577PAN_1756PAN_2636PAN_5334

UPenn

CD Non-IBDPAN_4600PAN_3404PAN_2405PAN_4857PAN_2549PAN_1946PAN_5317PAN_4139PAN_5285PAN_194PAN_5164PAN_5526PAN_4439PAN_1374PAN_2220PAN_617PAN_1756PAN_1932PAN_1642PAN_4841PAN_5072PAN_3091PAN_3824PAN_1403PAN_5448PAN_4787PAN_3662PAN_3524PAN_2232PAN_4968PAN_5334PAN_1370PAN_4653PAN_5372PAN_4471PAN_454PAN_5137PAN_2321PAN_4704PAN_3737PAN_1987PAN_5163PAN_3965PAN_4872PAN_4789PAN_709PAN_2636PAN_4658PAN_2474PAN_575PAN_5040PAN_5089PAN_2584PAN_5365PAN_719PAN_4304PAN_3769PAN_5131PAN_3577PAN_4950PAN_5054

UPenn

CD Non-IBD

Fru

ctose

,g

ala

ctito

l-re

late

dL

on

g-c

ha

infa

tty

aci

d-r

ela

ted

Ma

nn

ose

-re

late

d

PRISM

CD UC Non-IBD

Fru

ctose

,g

ala

ctito

l-re

late

dL

on

g-c

ha

infa

tty

aci

d-r

ela

ted

Ma

nn

ose

-re

late

d

RPKM

0

20

40

60

80

100

Fig. 4C

Fig. 4D

Old New

fig. S13. Fig. 2 and 4 expanded to show gene IDs.

References and Notes 1. I. Nasidze, J. Li, D. Quinque, K. Tang, M. Stoneking, Global diversity in the human salivary

microbiome. Genome Res. 19, 636–643 (2009). doi:10.1101/gr.084616.108 Medline

2. S. P. Humphrey, R. T. Williamson, A review of saliva: Normal composition, flow, and function. J. Prosthet. Dent. 85, 162–169 (2001). doi:10.1067/mpr.2001.113778 Medline

3. H. Seedorf, N. W. Griffin, V. K. Ridaura, A. Reyes, J. Cheng, F. E. Rey, M. I. Smith, G. M. Simon, R. H. Scheffrahn, D. Woebken, A. M. Spormann, W. Van Treuren, L. K. Ursell, M. Pirrung, A. Robbins-Pianka, B. L. Cantarel, V. Lombard, B. Henrissat, R. Knight, J. I. Gordon, Bacteria from diverse habitats colonize and compete in the mouse gut. Cell 159, 253–266 (2014). doi:10.1016/j.cell.2014.09.008 Medline

4. D. Gevers, S. Kugathasan, L. A. Denson, Y. Vázquez-Baeza, W. Van Treuren, B. Ren, E. Schwager, D. Knights, S. J. Song, M. Yassour, X. C. Morgan, A. D. Kostic, C. Luo, A. González, D. McDonald, Y. Haberman, T. Walters, S. Baker, J. Rosh, M. Stephens, M. Heyman, J. Markowitz, R. Baldassano, A. Griffiths, F. Sylvester, D. Mack, S. Kim, W. Crandall, J. Hyams, C. Huttenhower, R. Knight, R. J. Xavier, The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 15, 382–392 (2014). doi:10.1016/j.chom.2014.02.005 Medline

5. C. A. Lozupone, M. Li, T. B. Campbell, S. C. Flores, D. Linderman, M. J. Gebert, R. Knight, A. P. Fontenot, B. E. Palmer, Alterations in the gut microbiota associated with HIV-1 infection. Cell Host Microbe 14, 329–339 (2013). doi:10.1016/j.chom.2013.08.006 Medline

6. I. Vujkovic-Cvijin, R. M. Dunham, S. Iwai, M. C. Maher, R. G. Albright, M. J. Broadhurst, R. D. Hernandez, M. M. Lederman, Y. Huang, M. Somsouk, S. G. Deeks, P. W. Hunt, S. V. Lynch, J. M. McCune, Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism. Sci. Transl. Med. 5, 193ra91 (2013). doi:10.1126/scitranslmed.3006438 Medline

7. N. Qin, F. Yang, A. Li, E. Prifti, Y. Chen, L. Shao, J. Guo, E. Le Chatelier, J. Yao, L. Wu, J. Zhou, S. Ni, L. Liu, N. Pons, J. M. Batto, S. P. Kennedy, P. Leonard, C. Yuan, W. Ding, Y. Chen, X. Hu, B. Zheng, G. Qian, W. Xu, S. D. Ehrlich, S. Zheng, L. Li, Alterations of the human gut microbiome in liver cirrhosis. Nature 513, 59–64 (2014). doi:10.1038/nature13568 Medline

8. Y. Chen, F. Ji, J. Guo, D. Shi, D. Fang, L. Li, Dysbiosis of small intestinal microbiota in liver cirrhosis and its association with etiology. Sci. Rep. 6, 34055 (2016). doi:10.1038/srep34055 Medline

9. C. L. Sears, W. S. Garrett, Microbes, microbiota, and colon cancer. Cell Host Microbe 15, 317–328 (2014). doi:10.1016/j.chom.2014.02.007 Medline

10. E. S. Snitkin, A. M. Zelazny, P. J. Thomas, F. Stock, NISC Comparative Sequencing Program Group, D. K. Henderson, T. N. Palmore, J. A. Segre, Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci. Transl. Med. 4, 148ra116 (2012). doi:10.1126/scitranslmed.3004129 Medline

11. K. E. Holt, H. Wertheim, R. N. Zadoks, S. Baker, C. A. Whitehouse, D. Dance, A. Jenney, T. R. Connor, L. Y. Hsu, J. Severin, S. Brisse, H. Cao, J. Wilksch, C. Gorrie, M. B. Schultz, D. J. Edwards, K. V. Nguyen, T. V. Nguyen, T. T. Dao, M. Mensink, V. L. Minh, N. T. K. Nhu, C. Schultsz, K. Kuntaman, P. N. Newton, C. E. Moore, R. A. Strugnell, N. R. Thomson, Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc. Natl. Acad. Sci. U.S.A. 112, E3574–E3581 (2015). doi:10.1073/pnas.1501049112 Medline

12. E. G. Pamer, Resurrecting the intestinal microbiota to combat antibiotic-resistant pathogens. Science 352, 535–538 (2016). doi:10.1126/science.aad9382 Medline

13. H. Chu, A. Khosravi, I. P. Kusumawardhani, A. H. K. Kwon, A. C. Vasconcelos, L. D. Cunha, A. E. Mayer, Y. Shen, W.-L. Wu, A. Kambal, S. R. Targan, R. J. Xavier, P. B. Ernst, D. R. Green, D. P. B. McGovern, H. W. Virgin, S. K. Mazmanian, Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease. Science 352, 1116–1120 (2016). doi:10.1126/science.aad9948 Medline

14. X. C. Morgan, T. L. Tickle, H. Sokol, D. Gevers, K. L. Devaney, D. V. Ward, J. A. Reyes, S. A. Shah, N. LeLeiko, S. B. Snapper, A. Bousvaros, J. Korzenik, B. E. Sands, R. J. Xavier, C. Huttenhower, Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 13, R79 (2012). doi:10.1186/gb-2012-13-9-r79 Medline

15. K. Chen, J. P. McAleer, Y. Lin, D. L. Paterson, M. Zheng, J. F. Alcorn, C. T. Weaver, J. K. Kolls, Th17 cells mediate clade-specific, serotype-independent mucosal immunity. Immunity 35, 997–1009 (2011). doi:10.1016/j.immuni.2011.10.018 Medline

16. H. Xiong, J. W. Keith, D. W. Samilo, R. A. Carter, I. M. Leiner, E. G. Pamer, Innate lymphocyte/Ly6Chi monocyte crosstalk promotes Klebsiella pneumoniae clearance. Cell 165, 679–689 (2016). doi:10.1016/j.cell.2016.03.017 Medline

17. A. R. Records, The type VI secretion system: A multipurpose delivery system with a phage-like machinery. Mol. Plant Microbe Interact. 24, 751–757 (2011). doi:10.1094/MPMI-11-10-0262 Medline

18. S. Fukuda, H. Toh, K. Hase, K. Oshima, Y. Nakanishi, K. Yoshimura, T. Tobe, J. M. Clarke, D. L. Topping, T. Suzuki, T. D. Taylor, K. Itoh, J. Kikuchi, H. Morita, M. Hattori, H. Ohno, Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 469, 543–547 (2011). doi:10.1038/nature09646 Medline

19. A. N. Thorburn, L. Macia, C. R. Mackay, Diet, metabolites, and “Western-lifestyle” inflammatory diseases. Immunity 40, 833–842 (2014). doi:10.1016/j.immuni.2014.05.014 Medline

20. K. Chen, M. Goodwin, J. McAleer, N. Nguyen, E. Way, J. Kolls, Recombinant outer membrane protein: A potential candidate for Th17 based vaccine against Klebsiella pneumoniae. (VAC7P.967). J. Immunol. 192, 141.12 (2014).

21. B. H. Kim, J. D. Chee, C. J. Bradfield, E.-S. Park, P. Kumar, J. D. MacMicking, Interferon-induced guanylate-binding proteins in inflammasome activation and host defense. Nat. Immunol. 17, 481–489 (2016). doi:10.1038/ni.3440 Medline

22. S. E. Winter, C. A. Lopez, A. J. Bäumler, The dynamics of gut-associated microbial communities during inflammation. EMBO Rep. 14, 319–327 (2013). doi:10.1038/embor.2013.27 Medline

23. Y. Haberman, T. L. Tickle, P. J. Dexheimer, M.-O. Kim, D. Tang, R. Karns, R. N. Baldassano, J. D. Noe, J. Rosh, J. Markowitz, M. B. Heyman, A. M. Griffiths, W. V. Crandall, D. R. Mack, S. S. Baker, C. Huttenhower, D. J. Keljo, J. S. Hyams, S. Kugathasan, T. D. Walters, B. Aronow, R. J. Xavier, D. Gevers, L. A. Denson, Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature. J. Clin. Invest. 124, 3617–3633 (2014). doi:10.1172/JCI75436 Medline

24. Y. Taur, E. G. Pamer, The intestinal microbiota and susceptibility to infection in immunocompromised patients. Curr. Opin. Infect. Dis. 26, 332–337 (2013). doi:10.1097/QCO.0b013e3283630dd3 Medline

25. A. Davin-Regli, J. M. Pagès, Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front. Microbiol. 6, 392 (2015). doi:10.3389/fmicb.2015.00392 Medline

26. S. Mondot, S. Kang, J. P. Furet, D. Aguirre de Carcer, C. McSweeney, M. Morrison, P. Marteau, J. Doré, M. Leclerc, Highlighting new phylogenetic specificities of Crohn’s disease microbiota. Inflamm. Bowel Dis. 17, 185–192 (2011). doi:10.1002/ibd.21436 Medline

27. S. M. Diene, V. Merhej, M. Henry, A. El Filali, V. Roux, C. Robert, S. Azza, F. Gavory, V. Barbe, B. La Scola, D. Raoult, J.-M. Rolain, The rhizome of the multidrug-resistant Enterobacter aerogenes genome reveals how new “killer bugs” are created because of a sympatric lifestyle. Mol. Biol. Evol. 30, 369–383 (2013). doi:10.1093/molbev/mss236 Medline

28. J. D. Lewis, E. Z. Chen, R. N. Baldassano, A. R. Otley, A. M. Griffiths, D. Lee, K. Bittinger, A. Bailey, E. S. Friedman, C. Hoffmann, L. Albenberg, R. Sinha, C. Compher, E. Gilroy, L. Nessel, A. Grant, C. Chehoud, H. Li, G. D. Wu, F. D. Bushman, Inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric Crohn’s disease. Cell Host Microbe 18, 489–500 (2015). doi:10.1016/j.chom.2015.09.008 Medline

29. N. R. Shin, T. W. Whon, J. W. Bae, Proteobacteria: Microbial signature of dysbiosis in gut microbiota. Trends Biotechnol. 33, 496–503 (2015). doi:10.1016/j.tibtech.2015.06.011 Medline

30. H. Tiwana, R. S. Walmsley, C. Wilson, J. Y. Yiannakou, P. J. Ciclitira, A. J. Wakefield, A. Ebringer, Characterization of the humoral immune response to Klebsiella species in inflammatory bowel disease and ankylosing spondylitis. Br. J. Rheumatol. 37, 525–531 (1998). doi:10.1093/rheumatology/37.5.525 Medline

31. T. Rashid, A. Ebringer, C. Wilson, The role of Klebsiella in Crohn’s disease with a potential for the use of antimicrobial measures. Int. J. Rheumatol. 2013, 610393 (2013). doi:10.1155/2013/610393 Medline

32. W. S. Garrett, C. A. Gallini, T. Yatsunenko, M. Michaud, A. DuBois, M. L. Delaney, S. Punit, M. Karlsson, L. Bry, J. N. Glickman, J. I. Gordon, A. B. Onderdonk, L. H. Glimcher, Enterobacteriaceae act in concert with the gut microbiota to induce spontaneous and maternally transmitted colitis. Cell Host Microbe 8, 292–300 (2010). doi:10.1016/j.chom.2010.08.004 Medline

33. A. Ebringer, T. Rashid, H. Tiwana, C. Wilson, A possible link between Crohn’s disease and ankylosing spondylitis via Klebsiella infections. Clin. Rheumatol. 26, 289–297 (2007). doi:10.1007/s10067-006-0391-2 Medline

34. H. S. Said, W. Suda, S. Nakagome, H. Chinen, K. Oshima, S. Kim, R. Kimura, A. Iraha, H. Ishida, J. Fujita, S. Mano, H. Morita, T. Dohi, H. Oota, M. Hattori, Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers. DNA Res. 21, 15–25 (2014). doi:10.1093/dnares/dst037 Medline

35. K. W. Lee, S. Periasamy, M. Mukherjee, C. Xie, S. Kjelleberg, S. A. Rice, Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm. ISME J. 8, 894–907 (2014). doi:10.1038/ismej.2013.194 Medline

36. W. Huang, B. T. Sherman, R. A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009). Medline

37. B. D. Ondov, T. J. Treangen, P. Melsted, A. B. Mallonee, N. H. Bergman, S. Koren, A. M. Phillippy, Mash: Fast genome and metagenome distance estimation using MinHash. Genome Biol. 17, 132 (2016). doi:10.1186/s13059-016-0997-x Medline

38. S. Brisse, V. Passet, A. B. Haugaard, A. Babosan, N. Kassis-Chikhani, C. Struve, D. Decré, wzi gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J. Clin. Microbiol. 51, 4073–4078 (2013). doi:10.1128/JCM.01924-13 Medline

39. Y. J. Pan, T.-L. Lin, Y.-H. Chen, C.-R. Hsu, P.-F. Hsieh, M.-C. Wu, J.-T. Wang, Capsular types of Klebsiella pneumoniae revisited by wzc sequencing. PLOS ONE 8, e80670 (2013). doi:10.1371/journal.pone.0080670 Medline