DOI: 10.5061/dryad.2jm63xt21
title: TNF signaling maintains local restriction of bacterial founder populations in intestinal and systemic sites during oral Yersinia infection

description: Enteroinvasive bacterial pathogens are responsible for an enormous
 worldwide disease burden that critically affects the young and
 immunocompromised. Yersinia pseudotuberculosisis a Gram-negative enteric
 pathogen, closely related to the plague agent Y. pestis, that colonizes
 intestinal tissues, induces the formation of pyogranulomas along the
 intestinal tract, and disseminates to systemic organs following oral
 infection of experimental rodents. Prior studies proposed that systemic
 tissues were colonized by a pool of intestinal replicating bacteria
 distinct from populations within Peyer’s patches and mesenteric lymph
 nodes. Whether bacteria within intestinal pyogranulomas serve as the
 source for systemic dissemination, and the relationship between bacterial
 populations within different tissue sites, is poorly defined. Moreover,
 the factors regulating Yersinia colonization and dissemination are
 poorly understood. Here, we demonstrate, using Sequence Tag-based
 Analysis of Microbial Populations in R (STAMPR), that remarkably small
 founder populations independently colonize intestinal and systemic
 tissues. Notably, intestinal pyogranulomas contain clonal populations of
 bacteria that are restricted and do not spread to other tissues. However,
 populations of Yersinia are shared among systemic organs and the blood,
 suggesting that systemic dissemination occurs via hematogenous spread.
 Finally, we demonstrate that TNF signaling is a key contributor to the
 bottlenecks limiting both tissue colonization and lymphatic dissemination
 of intestinal bacterial populations. Altogether, this study reveals
 previously undescribed aspects of the infection dynamics of enteric
 bacterial pathogens.
description: <strong>Sample harvesting</strong>
 Samples were harvested as described in Extended Methods
 (Supplemental Information). All samples had 100uL
 serially diluted tenfold in PBS, plated on LB agar supplemented with 2
 μg/ml triclosan and 100 mg/mL kanamycin, and incubated for two days at
 room temperature. Dilutions of each sample were plated in triplicate and
 expressed as the mean CFU per gram or per biopsy. Remaining 900uL of
 homogenized samples were plated on 15 cm dishes with LB agar supplemented
 with 2 μg/ml triclosan (irgasan) and 100 mg/mL kanamycin and incubated for
 two days at room temperature. <strong>STAMP
 sample processing</strong> <em>Y.
 ptb</em> colonies were washed off plates, collected in PBS with 25%
 glycerol, diluted in water, and boiled for 15 minutes at 95°C. The
 barcode-containing region was amplified from the genome using custom
 forward and reverse primers(Campbell et al., 2023; Holmes et al., 2025;
 Hotinger et al., 2025). Sequence tag-based analysis of microbial
 populations (STAMP) was performed as previously described(Holmes et al.,
 2025). Any additional information required to reanalyze the data reported
 in this paper is available from the lead contact upon request.
 <strong>Statistics</strong>
 Graphing and statistical analyses of data were performed using
 Prism 9 software (GraphPad). Statistical significance was determined using
 the statistical tests indicated in each figure legend. Differences were
 considered statistically significant if the <em>P </em>value
 was less than or equal to 0.05.
description: # TNF signaling maintains local restriction of bacterial founder
 populations in intestinal and systemic sites during oral Yersinia
 infection Dataset DOI: [10.5061/dryad.2jm63xt21](10.5061/dryad.2jm63xt21)
 ## Description of the data and file structure ### Associated Figure
 Legends Some data is organized into folders by figure where multiple
 datasets from related experiments were presented together. In order to
 understand and analyze the data, here we provide brief descriptions of the
 figures from the related manuscript. **Figure 1. Bacterial tissue burdens
 are driven by extensive replication of small numbers of initial
 founders.** (A) Bacterial burdens in Peyer’s patch (PP), mesenteric lymph
 node (MLN), spleen, and liver tissues at day 5 post-infection. (B)
 Bacterial burdens in small intestinal pyogranuloma (PG+) and adjacent
 non-granulomatous (PG-) tissue isolated day 5 post-infection. Each circle
 represents the total CFU of 5-10 pooled punch biopsies divided by number
 of pooled punch biopsies from one mouse. (C) Founding population (Ns) in
 indicated tissues at day 5 post-infection. (D) Founding population (Ns) in
 small intestinal PG+ and PG- tissue isolated day 5 post-infection. Each
 circle represents the total Ns of 5-10 pooled punch biopsies divided by
 number of pooled punch biopsies from one mouse. (E) Y. ptb bacterial
 burden in flushed small intestinal contents (IC) at day 5 post-infection.
 (F) Founding population (Ns) in flushed IC at day 5 post-infection.
 Samples labeled liver are showing the median lobe as representative of all
 liver lobes. For all data, circles represent one mouse, bars are mean ±
 standard error of the mean (SEM), and data are pooled from three
 experiments. Statistical significance was only calculated on B,D using
 Mann-Whitney tests and **** p&lt;0.0001. **Figure 2. Peyer’s patches and
 intestinal pyogranulomas contain distinct bacterial populations.** (A)
 Bacterial burdens and (B) founding population (Ns) in small intestinal PP,
 PG+, and PG- tissue isolated day 5 post-infection. Each circle represents
 one biopsy.  Bars represent (A) mean ± SEM or (B) median ± 95% CI. Pooled
 data from four mice from one experiment. (C) Frequency of barcodes per
 biopsy for mouse 1. Each bar represents one biopsy, and each color
 represents one barcode. (D) Similarity between Y. ptb populations in each
 biopsy as assessed by chord distance (CD) for mouse 1, depicted as (TOP)
 samples ordered by location along small intestine or (BOTTOM) samples
 ordered by biopsy type. Red boxes highlight two examples of sharing
 dynamics where PP share with adjacent PG-, but not with adjacent PG+.
 (n=4, one experiment, equivalent data of mice 2-4 is depicted in Fig
 S2E-J). (E) Histograms showing the frequency of binned CD values for each
 comparison: (E) PP vs PP (blue), PP vs PG- (cyan), and PP vs PG+(magenta);
 (F) PG+ vs PG+ (red) and PG+ vs PG- (yellow); and (G) PG- vs PG-(green).
 For E-G, color-coding of comparisons is diagrammed by triple Venn diagram
 with specific comparisons highlighted in the figure key, bin width = 0.1,
 and the center of bins are shown on x-axis. Data is cumulative from 4
 mice, one experiment. **Figure 3. Bacterial populations in the spleen and
 liver are distinct from those in the intestine.** (A) Similarity between
 Y. ptb populations in each sample as assessed by chord distance (CD) for
 one representative mouse. (B) CD between spleen and indicated samples.
 (C-D) Prevalence of barcodes from (C) the spleen in other organs
 (FRDorgan-spleen) or (D) from various organs in the spleen
 (FRDspleen-organ). (E) Fraction of barcodes from the spleen that are not
 found in other sites outside of the spleen or liver lobes. All data
 collected at day 5 post-infection, each circle represents one mouse, n=10,
 pooled data from three independent experiments, where bars are mean ± SEM
 and violin plots indicate median ± interquartile range. Statistical
 significance was calculated using one way ANOVA with post-hoc Dunn’s
 Multiple Comparison Test, where ns = not significant and * p&lt;0.05.
 **Figure 4. Blood is a conduit for systemic Y. ptb populations.** (A) CD
 between the MLN and indicated samples. (B) Bacterial burdens and (C)
 Founding population (Ns) in blood; blood samples with no CFU are
 represented as 0.1 CFU. (D) Log-log regression with Pearson’s correlation
 of fit between Ns and CFU measured in blood. (C-D) Each circle represents
 one mouse with detectible burden of *Y. ptb *in blood. (E) CD between the
 blood and indicated samples. Samples labeled liver are showing CD
 comparisons to the median lobe as representative of all liver lobes. All
 data were collected at day 5 post-infection, from three-four independent
 experiments (except B, which has pooled data from seven experiments),
 where each circle represent one mouse unless indicated otherwise and bars
 are mean ± SEM. **Fig. 5. TNFR1 contributes to Y. ptb infection bottleneck
 and MLN colonization.** (A) Bacterial burdens and (B) founding population
 (Ns) in small intestinal PP, PG+, and PG- biopsies at day 5
 post-infection, where each symbol represents the total of 5-10 pooled
 punch biopsies divided by the number of biopsies from one mouse. (C)
 Bacterial burdens and (D) founding population (Ns) in whole blood. (E)
 Bacterial burdens and (F) founding populations (Ns) in systemic tissues:
 mesenteric lymph nodes (MLN), spleen, and liver (median lobe). (G-I) Chord
 Distance (CD) between (G) PG+, (H) spleen, or (I) MLN and the indicated
 systemic (MLN, spleen, median liver lobe, blood) and intestinal (PP and
 PG-) tissues. All data collected on day 5 post-infection and are pooled
 from three independent experiments. (J-K) H&E-stained
 paraffin-embedded mesenteric lymph node sections from naïve and
 Y.ptb-infected WT and Tnfr1−/− mice at day 5 after infection were used for
 (J) histopathological scoring and (K) imaging. (J) Each mouse was scored
 between 0 and 4 (minimal to extensive) for the metrics shown in Fig.
 S4E-G. Scores for each mouse were added together to obtain the aggregate
 score shown. (K) Naïve mesenteric lymph nodes from WT mice (LEFT), but not
 Tnfr1−/− mice (RIGHT), have lymphoid follicles in the cortex (marked by
 arrowheads). White scale bars = 100 μm. Infected mesenteric lymph nodes
 (BOTTOM) show pyogranulomatous lesions in WT mice (marked by asterisks)
 and necrosis in Tnfr1−/− mice (marked by arrows). Black scale bars = 200
 μm. Histological data are from two independent experiments with
 representative images shown. Each symbol represents one mouse unless
 otherwise indicated, bars are mean ± SEM. Statistical significance was
 calculated using multiple Mann-Whitney tests and ns = not significant, *
 p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001.  **Figure S1. Validation of the
 Y. ptb barcode library and impact of antibiotic pre-treatment on barcoded
 Y.ptb dissemination.** (A) The ability to determine the size of the
 founding population from STAMP libraries was validated in culture by
 comparing the number of plated colonies (CFU; known number of founders) to
 the size of the founding population following STAMPR analysis of those
 plated populations (Ns) for 3 cultures across 9 doses. (B) Bacterial
 burdens and (C) founding population (Ns) in stool isolated 1-, 6-, or
 12-hours post-infection. Pooled data from four mice from one experiment.
 (D) Graphical representation of the experimental design for antibiotic
 (ABX) pretreatment: C57BL/6J mice were given either a cocktail of ABX or
 control sterile lab drinking water ad libitum for 7 days and returned to
 normal drinking water one day prior to infection. At day 0, mice were
 inoculated with 2x108 CFU Y. ptb library via oral gavage. Mice were
 euthanized 5 days following the inoculation and Y. ptb populations were
 assessed. (E) Bacterial burden and (F) founding population (Ns) in flushed
 small intestinal contents. (G) Bacterial burdens and (H) founding
 population (Ns) in small intestinal PP, PG+, and PG- biopsies. Each circle
 represents the total CFU of 5-10 pooled punch biopsies divided by number
 of pooled punch biopsies from one mouse. (I) Bacterial burden and (J)
 founding population (Ns) in MLN, spleen, and liver. Unless otherwise
 indicated, each circle represents one mouse, and all graphs are pooled
 data from three independent experiments.  For bar graphs, bars represent
 mean ± SEM, and statistical significance was determined using Mann-Whitney
 tests: ns = not significant, * p&lt;0.05, ** p&lt;0.01, **** p&lt;0.0001.
 **Figure S2. Intestinal biopsy maps, analysis of barcodes in mouse
 intestine, and pooled comparisons of chord distance.** (A-D) Map showing a
 representative layout of individually harvested and processed tissue
 biopsies from the small intestine, cecum, and colon of mice 1-4 on day 5
 post-infection. (E-G) Frequency of barcodes per biopsy for mice 2-4 (mouse
 1 is depicted in Fig. 1C), where each bar represents one biopsy, and each
 color represents one barcode. (H-J) Similarity between Y. ptb populations
 in each biopsy as assessed by chord distance (CD) for mice 2-4 (mouse 1 is
 depicted in Fig. 1D), where (LEFT) data is organized by biopsy type and
 (RIGHT) data is ordered by location along gastrointestinal tract. (K) CD
 between biopsies within the indicated small intestinal region. Each circle
 represents one comparison between biopsies within the indicated region for
 that mouse. (L) Proportion of PG+ biopsies that share or do not share with
 PP or PG- biopsies in the same mouse. Sharing was determined by presence
 of single PG+ founder in other biopsies or, for PG+ containing more than
 one founder, the most abundant barcode was used to determine sharing.
 (M-N) CD between the indicated biopsy types. Each circle represents one
 comparison between the (M) same or (N) different biopsy types as
 indicated, where biopsies are matched within mouse. Unless otherwise
 indicated, for all data bars represent mean ± SEM, (pooled for n=4 mice,
 one experiment collected at 5 days post-infection). Statistical
 significance was determined using one-way ANOVA with post-hoc Dunn’s
 Multiple Comparison Test, where ns = not significant and **** p&lt;0.0001.
 **Figure S3. Populations of Y. ptb in spleen and liver are similar to each
 other with one highly abundant clone.** (A) CD between the spleen and
 gastrointestinal tract organ tissues. (B) CD between the spleen and
 individual liver lobes. (C) Frequency of barcodes per tissue for one
 representative mouse. Each bar represents one sample, and each color
 represents one barcode. (D) Frequency of most abundant barcode per sample.
 All samples collected at 5 days post-infection. Data are pooled from three
 to five experiments, each circle represents one mouse, bars are mean ± SEM
 unless otherwise indicated. **Figure S4. TNFR1 signaling affects bacterial
 colony containment in mesenteric lymph nodes but not clone expansion or
 lymphadenitis.** (A) Bacterial burdens and (B) founding populations (Ns)
 in flushed small intestinal contents at day 5 post-infection. (C-F)
 **Bacterial burden per founder, as determined by CFU/Ns, in (C) flushed
 small intestinal contents, (D) small intestinal PP, PG+, and PG- biopsies,
 (E) whole blood, and (F) indicated systemic tissues at day 5
 post-infection. Liver samples show data from the median lobe as
 representative of all liver lobes. Pooled data from three independent
 experiments. (G-I) H&E-stained paraffin-embedded mesenteric lymph node
 sections from naïve and Y.ptb-infected WT and Tnfr1−/− mice at day 5 after
 infection were scored between 0 and 4 (minimal to extensive) for the
 metrics shown. Histological data are from two independent experiments.
 Each symbol represents one mouse, and bars are mean ± SEM. Statistical
 significance was determined using multiple Mann-Whitney tests and ns = not
 significant, * p&lt;0.05, ** p&lt;0.01. ### Files and variables #### File:
 Frequencyplotanalysis.zip **Description:** This file contains .xlsx files
 with processed data from multiple FrequenciesWithoutNoise.csv files. We
 have left this data as .xlsx to keep related data together in multiple
 tabs and maintain formatting that makes the data more readable. However,
 this formatting is not required for accessibility or reanalysis. This
 processed data was used to describe and depict the frequency of barcodes
 in samples displayed in Fig. 2, Fig. 3, Fig. S2, and Fig. S3. #### File:
 RawCFU.zip **Description:** This folder contains raw counts of bacterial
 colonies used to determine the CFU and CFU/g for harvested mouse tissues.
 Files are organized in subfolders by figure. We have left this data as
 .xlsx to maintain text formatting that makes the data more readable and to
 provide access to formulas used in calculating CFU and CFU/g. This
 formatting is not required for accessibility or reanalysis. Each file
 lists samples in rows containing (left to right): experiment name, sample
 (key provided in file), group (genotype or treatment group), colony counts
 from triplicate spot plating, the dilution that was counted, the tube
 weights pre-and post-tissue collection, calculated CFU and CFU/g, the
 number of colonies on the plate collected for sequencing (if countable).
 Blood samples also have the volume of blood collected listed.
 Additionally, the number of intestinal biopsies of Peyer’s patches,
 intestinal pyogranulomas, and adjacent nongranulomatous tissue is listed
 for each mouse. These files were used to generate CFU/g or CFU/biopsy
 plots and CFU tables for STAMPR input. #### File: Frequencies.zip
 **Description:** This file contains .csv files with the barcode
 frequencies as determined by analysis of raw sequencing reads and removal
 of barcode hopping. These files were used as input into the STAMPR
 analysis code provided and as previously described (Holmes et al., 2025;
 Hullahalli et al., 2021). Each sequencing batch of 64 samples was labeled
 in order with “spXX”. This batch ID identifies all downstream files
 generated through STAMPR analysis. #### File: STAMPRcode.zip
 **Description:** This file contains R scripts used for STAMPR analysis
 after index hopping correction. getFP.R determines founding population
 estimates using the frequency table and the CFU table as inputs.
 MajorityDistance.R calculates genetic distance metrics referred to as
 “Chord distance” in our work. #### File: STAMPRinputs.zip
 **Description:** This folder contains .csv files input into the provided
 STAMPR analysis pipeline. Each analysis has a CFU table that was fed into
 the getFP.R script to determine founding population estimates.
 Subsequently, GD_metadata files were used to calculate genetic distance
 metrics using the MajorityDistance R script. Samples can be identified by
 the BioSample ID that lists “experiment#_Sample” (e.g., “SP051_S1”). Files
 are organized into subfolders based on the relevant figure. #### File:
 MappedBiopsies.zip **Description:** This file contains original images of
 hand-drawn maps made when harvesting individual intestinal biopsies in
 Fig. 2 and Fig. S2. #### File: STAMPRoutputs.zip **Description:** This
 file contains .csv files generated by the STAMPR analysis pipeline. Each
 file is identified by the sequencing batch ID “spXX”. Occasionally,
 samples were sequenced in different batches. Thus, some files have been
 modified to include samples from multiple sequencing batches. In these
 cases, the experiment number “SP0XX” is used to identify the samples in
 the file. Analysis of the files in Frequencies.zip generated each
 NsNb.csv, FrequenciesWithoutNoise.csv, GD.csv, RD.csv, and FRD.csv file.
 #### File: Histology.zip **Description:** This file contains raw data
 relevant to MLN histology shown in Fig 5J,K and Fig S4G-I. The two .xlsx
 files are from two independent experiments where blinded analysis of
 histological sections was done by the comparative pathology core. We have
 left this data as .xlsx to keep the full analysis together in multiple
 tabs to aid in comprehension and any future reanalysis. The tab labeled
 “Heatmap of Lesions” has scores plotted in Fig 5J and Fig S4G-I.
 Formatting, as provided by the comparative pathology core, was left on for
 clarity and visual assessment. However, this formatting is not required
 for accessibility or reanalysis. Unmodified .tif images of H&E-stained
 histological sections are present for each of the representative sections
 shown in Fig 5K. #### Code/software No Code or Software other than
 Microsoft Excel is needed to view this data. To reanalyze, R scripts are
 provided for STAMPR analysis. #### Access information All raw data were
 generated from new experiments described in the manuscript associated with
 this dataset. Please disregard any other highlighting or colors, as they
 were used for personal reference.  Abbreviations: WT = wild-type, ABX =
 antibiotics-treated, S = spleen, L1 = left liver lobe, L2 = median liver
 lobe,  L3 = right liver lobe, L4 = caudate liver lobe, M = mesenteric
 lymph node, B = blood, P = Peyer’s patches, + = intestinal pyogranuloma
 biopsies, - = intestinal biopsies that do not contain pyogranulomas, IC =
 intestinal contents, CFU = colony forming units, STAMPR = Sequence
 tag-based analysis of microbial populations in R