DNA methylation is related to bacterial burden and differs among tissues in infected house sparrows
Data files
Apr 30, 2026 version files 141.85 KB
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DNAm_blood_finalSB.csv
47.26 KB
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DNAm_blood_imputed.csv
47.11 KB
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DNAm_euthanasia_data.csv
23.82 KB
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DNAm_SB_data_summary_full.csv
7.93 KB
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README.md
15.72 KB
Sep 11, 2026 version files 147.53 KB
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DNAm_blood_finalSB.csv
47.26 KB
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DNAm_euthanasia_data.csv
23.82 KB
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DNAm_SB_data_summary_full.csv
7.93 KB
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hosp_tissues_DNAm_euthgex.csv
51.82 KB
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README.md
16.70 KB
Abstract
Epigenetic mechanisms, particularly DNA methylation, are environmentally sensitive and flexible regulators of gene expression that may facilitate responses to infection. We investigated whether DNA methylation across five CpGs within the putative promoter of Toll-like Receptor-04, a gene important for immune surveillance and response to Gram-negative bacteria, was associated with individual resistance to pathogenic Salmonella enterica in house sparrows (Passer domesticus). House sparrows are a globally distributed songbird with introduced ranges on five continents and previous research supports that DNA methylation is related to TLR4 expression. We quantified DNA methylation in blood across infection, and in liver, blood, and spleen at euthanasia. DNA methylation changed within 6 hours of exposure. Faster decreases in DNA methylation were associated with lower Salmonella burden, suggesting a role in control of bacterial infection. Changes in DNA methylation differed among CpGs, with the strongest declines found 1244 and 1089 bp upstream of the transcription start site. DNA methylation was positively correlated within individuals across tissues, supporting the use of blood as a general indicator of DNA methylation in inaccessible tissues. We also detected DNA methylation differences among tissues, with liver higher than blood and spleen. Specifically, liver DNA methylation was higher at CpGs 1, 2, and 3, while CpGs 4 and 5 were constitutively lower across tissues. This pattern reflects that CpG sites change DNA methylation discordantly, and that particular sites may have a larger role in the response to infection. Our results show DNA methylation in TLR4 is linked to infection outcomes of house sparrows.
Dataset DOI: 10.5061/dryad.3tx95x6x3
Description of the data and file structure
Our objective was to determine if DNA methylation in the putative promoter of TLR4 was related to the response to Salmonella infection for house sparrows. Using the same individuals as Sheldon et al. (2023), we compared DNA methylation temporally across the infection in blood samples and post-euthanasia among multiple tissues (i.e., liver, spleen, and blood).
We screened house sparrows from Tampa, FL, USA (n = 36) that had been used in a study involving experimental infection with Salmonella enterica (described in Sheldon et al., 2023). Individuals were classified as high EP or low EP and characterized for sex (adult female, adult male, and juvenile) and body mass (Sheldon et al., 2023). Salmonella burden was quantified from fecal samples across the experiment in Sheldon et al. (2023). Relativized gene expression of TLR4 in the proximal intestine, medial intestine, distal intestine, and caecum was quantified at euthanasia in Sheldon et al. (2023). To describe DNA methylation changes in birds, we used blood samples at capture (n = 28), 6-hours post-infection (n = 29), 1-day post-infection (n = 27), 6-days post-infection (n = 27), 9-days post-infection (n = 30), 12-days post-infection (n = 31), and at euthanasia, which was 14-days post-infection (n = 31). We also screened liver (n = 33) and spleen (n = 25) tissues for DNA methylation from the same individuals at euthanasia.
We edited and aligned sequences with MEGA version 11 (Tamura et al., 2021). We coded DNA methylation as binary for each CpG, 0 for not methylated and 1 for methylated.
First, we asked if the rate of change in DNA methylation was related to the rate of change in Salmonella burden over the infection period. We calculated an average proportion of DNA methylation across the 5 CpGs for each bird at each time during infection. We then calculated an individual rate of change in DNA methylation across the infection (Δ blood DNA methylation/day). We also calculated the rate of change in Salmonella burden (Δ Salmonella burden/day), the average Salmonella burden, and the final Salmonella burden. We compared the rate of change in DNA methylation to Salmonella burden estimates with Pearson correlations
We used Bayesian generalized linear mixed models (GLMM) to assess DNA methylation across the putative promoter of TLR4 and its effects on Salmonella burden.
We used multiple regressions to assess DNA methylation in blood, liver, and spleen across the putative promoter of TLR4 and its effects on gene expression of TLR4 in the proximal intestine, medial intestine, distal intestine, and caecum at euthanasia.
Files and variables
File: DNAm_SB_data_summary_full.csv
Description: DNAm_SB_data_summary_full is the bird-level summary reference file. One row per individual (n = 36). Contains biological stage, epigenetic potential (EP) category, body mass, average and final Salmonella burden, daily rates of change in Salmonella burden and DNA methylation, average and variance of DNA methylation per tissue at euthanasia, and per-CpG binary methylation values across all time points and tissues.
Raw data for Lauer et al. DNA methylation of house sparrows differs over time and among tissues and is related to reduced burden of an experimental infection. Missing values were indicated with "NA".
Variables
- BirdID (unique bird identifier)
- SEX (male, female, juvenile)
- Body Mass (g)
- Epigenetic Potential (CpG content of the putative promoter of TLR4; categorical high/low)
- Epigenetic potential (count of total CpGs in putative promoter of TLR4)
- Average Salmo Burden (average Salmonella burden during experimental infection, log10 genomic equivalents per day)
- Final Salmo Burden (Salmonella burden at euthanasia, log10 genomic equivalent)
- Daily Rate of Change SB (average daily rate of change in Salmonella burden, calculated as the delta between time points of collection; log10genomic equivalents)
- Average Liver DNAm (average liver DNA methylation across 5 CpG sites in putative promoter of TLR4 at euthanasia, calculated with binary DNA methylation data)
- Variance Liver DNAm (variance in liver DNA methylation across 5 CpG sites in putative promoter of TLR4 at euthanasia, calculated with binary DNA methylation data)
- Average Spleen DNAm (average spleen DNA methylation across 5 CpG sites in putative promoter of TLR4 at euthanasia, calculated with binary DNA methylation data)
- Variance Spleen DNAm (variance in spleen DNA methylation across 5 CpG sites in putative promoter of TLR4 at euthanasia, calculated with binary DNA methylation data)
- Average Blood DNAm (average whole blood DNA methylation across 5 CpG sites in putative promoter of TLR4 at euthanasia, calculated with binary DNA methylation data)
- Variance Blood DNAm (variance in whole blood DNA methylation across 5 CpG sites in putative promoter of TLR4 at euthanasia, calculated with binary DNA methylation data)
- BLOOD Change in DNAm per day (average daily rate of change in DNA methylation, calculated as the delta between time points of collection)
- Liver CG-05 (liver DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at euthanasia)
- Liver CG-04 (liver DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at euthanasia)
- Liver CG-03 (liver DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at euthanasia)
- Liver CG-02 (liver DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at euthanasia)
- Liver CG-01 (liver DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at euthanasia)
- Spleen CG-05 (liver DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at euthanasia)
- Spleen CG-04 (spleen DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at euthanasia)
- Spleen CG-03 (spleen DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at euthanasia)
- Spleen CG-02 (spleen DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at euthanasia)
- Spleen CG-01 (spleen DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at euthanasia)
- Blood CG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at euthanasia)
- Blood CG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at euthanasia)
- Blood CG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at euthanasia)
- Blood CG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at euthanasia)
- Blood CG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at euthanasia)
- Capture: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 upon capture)
- Capture: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 upon capture)
- Capture: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 upon capture)
- Capture: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 upon capture)
- Capture: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 upon capture)
- 6h: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at 6 hours post-infection)
- 6h: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at 6 hours post-infection)
- 6h: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at 6 hours post-infection)
- 6h: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at 6 hours post-infection)
- 6h: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at 6 hours post-infection)
- d01: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at day 1 post-infection)
- d01: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at day 1 post-infection)
- d01: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at day 1 post-infection)
- d01: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at day 1 post-infection)
- d01: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at day 1 post-infection)
- d06: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at day 6 post-infection)
- d06: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at day 6 post-infection)
- d06: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at day 6 post-infection)
- d06: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at day 6 post-infection)
- d06: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at day 6 post-infection)
- d09: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at day 9 post-infection)
- d09: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at day 9 post-infection)
- d09: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at day 9 post-infection)
- d09: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at day 9 post-infection)
- d09: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at day 9 post-infection)
- d12: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at day 12 post-infection)
- d12: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at day 12 post-infection)
- d12: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at day 12 post-infection)
- d12: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at day 12 post-infection)
- d12: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at day 12 post-infection)
- Euth: CpG-05 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 05 at euthanasia)
- Euth: CpG-04 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 04 at euthanasia)
- Euth: CpG-03 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 03 at euthanasia)
- Euth: CpG-02 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 02 at euthanasia)
- Euth: CpG-01 (whole blood DNA methylation presence/absence data coded as binary 1/0 for putative promoter of TLR4 CpG 01 at euthanasia)
File: DNAm_blood_finalSB.csv
Description: Long-format raw blood DNA methylation time series. One row per bird × CpG site × time point combination (1,155 rows total). Missing values listed as NA.
Variables
- BirdID: Individual bird identifier
- Stage: Biological stage (Female, Male, Juvenile)
- BodyMass: Body mass (g)
- EP: Epigenetic potential category (high, low)
- FinalSB: Final Salmonella burden at euthanasia (log₁₀ genomic equivalents)
- timeday: Collection time point in raw days (0, 0.25, 1, 6, 9, 12, 14)
- CGsite: CpG site identity (CG01–CG05; reference in models: CG01)
- DNAm: Binary DNA methylation state (0 = unmethylated, 1 = methylated; NA = missing, excluded listwise)
File: DNAm_euthanasia_data.csv
Description: Raw data long-format DNA methylation data at euthanasia across blood, liver, and spleen tissues. One row per bird × tissue × CpG site combination (540 rows total). Missing values listed as NA.
Variables
- BirdID: Individual bird identifier
- FinalSB: Final Salmonella burden (log₁₀ genomic equivalents)
- BodyMass: Body mass (g)
- Stage: Biological stage (Female, Male, Juvenile)
- EP: Epigenetic potential category (high, low)
- Tissue: Tissue type (blood, liver, spleen; reference in models: blood)
- SiteEuth: CpG site identity (CG01–CG05; reference in models: CG01)
- DNAm: Binary DNA methylation state (0 = unmethylated, 1 = methylated; NA = excluded listwise)
File: hosp_tissues_DNAm_euthgex.csv
Description: Raw data long-format DNA methylation data at euthanasia across blood, liver, and spleen tissues with relativized gene expression of TLR4 for proximal intestine, medial intestine, distal intestine, and caecum at euthanasia. One row per bird × tissue × CpG site combination (540 rows total). Missing values listed as NA.
Variables
- BirdID: Individual bird identifier
- FinalSB: Final Salmonella burden (log₁₀ genomic equivalents)
- SB_ROC: average daily rate of change in Salmonella burden, calculated as the delta between time points of collection (log10genomic equivalents)
- BodyMass: Body mass (g)
- Stage: Biological stage (Female, Male, Juvenile)
- EP: Epigenetic potential category (high, low)
- Tissue: Tissue type (blood, liver, spleen)
- SiteEuth: CpG site identity (CG01–CG05)
- DNAm: Binary DNA methylation state (0 = unmethylated, 1 = methylated; NA = excluded listwise)
- TLR4_RQ_proximal_intestine: relativized gene expression of TLR4 for proximal intestine at euthanasia, collected in Sheldon et al., 2023.
- TLR4_RQ_medial_intestine: relativized gene expression of TLR4 for medial intestine at euthanasia, collected in Sheldon et al., 2023.
- TLR4_RQ_distal_intestine: relativized gene expression of TLR4 for distal intestine at euthanasia, collected in Sheldon et al., 2023.
- TLR4_RQ_cecum: relativized gene expression of TLR4 for caecum at euthanasia, collected in Sheldon et al., 2023.
Code/software
Code and scripts for data analysis are available at DOI: 10.5281/zenodo.19872493 https://github.com/mellesselauer/Lauer_etal_hosp_TLR4_DNAm_Salmonella
Access information
Other publicly accessible locations of the data:
Full data, code, and scripts for data analysis are available at DOI: 10.5281/zenodo.19872493
https://github.com/mellesselauer/Lauer_etal_hosp_TLR4_DNAm_Salmonella
Salmonella burden, epigenetic potential, and gene expression of TLR4 in proximal intestine, medial intestine, distal intestine, and caecum data was published in the following sources:
- Sheldon, E., Zimmer, C., Hanson, H., Koussayer, B., Schrey, A., Reese, D., Wigley, P., Wedley, A.L. and Martin, L.B., 2023. High epigenetic potential protects songbirds against pathogenic Salmonella enterica infection. Journal of Experimental Biology, 226(13), p.jeb245475. Data available: https://doi.org/10.6084/m9.figshare.23596692
Sample Collection
We screened house sparrows from Tampa, FL, USA (n = 36) that had been used in a study involving experimental infection with Salmonella enterica (described in Sheldon et al., 2023). Individuals were classified as high EP or low EP and characterized for sex (adult female, adult male, and juvenile) and body mass (Sheldon et al., 2023). Salmonella burden was quantified from fecal samples across the experiment in Sheldon et al. (2023). Relativized gene expression of TLR4 in the proximal intestine, medial intestine, distal intestine, and caecum was quantified at euthanasia in Sheldon et al. (2023). To describe DNA methylation changes in birds, we used blood samples at capture (n = 28), 6-hours post-infection (n = 29), 1-day post-infection (n = 27), 6-days post-infection (n = 27), 9-days post-infection (n = 30), 12-days post-infection (n = 31), and at euthanasia, which was 14-days post-infection (n = 31). We also screened liver (n = 33) and spleen (n = 25) tissues for DNA methylation from the same individuals at euthanasia (Table S2).
Data Collection
We extracted DNA from each tissue sample with the Qiagen DNeasy kit (Qiagen, Valencia, CA). We deaminated unmethylated cytosines to thymines using the NEBNext® Enzymatic Methyl-seq conversion module (New England Biolabs, Ipswich, MA; Vaisvila et al., 2021). We then amplified the putative promoter region of TLR4 (Kilvitis et al., 2019) in 10 µl reactions with a final concentration of 1x PCR buffer, 2 mM of each dNTP, 1.5 mM MgCl2, 1.0 μg/μl BSA, 0.5 µM of each primer (Kilvitis et al., 2019), and 1 unit of Taq polymerase. We used a thermal profile consisting of an initial denaturing step at 94ºC for 3m, and then 30 cycles of 94ºC for 15s, 62ºC for 15s, and 72ºC for 1m with a final extension step at 72ºC for 10m. We cleaned reactions with ExoSAP-IT Express (ThermoFisher) and conducted Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, USA) on an ABI 3730xl DNA analyzer (Applied Biosystems, USA).
Data Analysis
We edited and aligned sequences with MEGA version 11 (Tamura et al., 2021). We coded DNA methylation as binary for each CpG, 0 for not methylated and 1 for methylated.
Changes after Apr 30, 2026: Edited README.md.
Deleted file DNAm_blood_imputed.csv - statistical methods were reconceptualized without a data imputation stage.
Uploaded file hosp_tissues_DNAm_euth_gex.csv.