Data from: Functional characterization of Paillotin: An immune peptide regulated by the Imd pathway with pathogen-specific roles in Drosophila immunity
Data files
Aug 01, 2025 version files 2.63 MB
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Data_and_code.zip
2.61 MB
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README.md
17.16 KB
Abstract
Insects, such as Drosophila melanogaster, rely on innate immune defenses to combat microbial threats. Antimicrobial peptides (AMPs) play an important role in limiting pathogen entry and colonization. Despite intensive research into the regulation and biochemical properties of AMPs, their exact significance in vivo has remained uncertain due to the challenges of mutating small genes. Fortunately, recent technologies have enabled the mutation of individual AMP genes, overcome previous obstacles, and opened new avenues for research. In this study, we characterized one novel host-defense peptide, Paillotin (IM18, CG33706), using loss-of-function mutants. Paillotin is an ancient host defense peptide of Diptera, regulated by the Imd pathway. Loss of Paillotin does not impact the activity of either the Imd or Toll pathways. Importantly, we found that Paillotin mutants are viable but exhibit increased susceptibility to specific infections, particularly Providencia burhodogranariea. Paillotin was further found to contribute synergistically to defense against P. burhodogranariea when combined with other AMPs. However, we did not detect direct microbicidal activity of Paillotin in vitro in our hands. Taken together, our findings identify Paillotin as a novel host defense peptide acting downstream of Imd signaling, advancing our understanding of the Drosophila antimicrobial response.
Dataset DOI: 10.5061/dryad.f4qrfj77k
Description of the data and file structure
This folder contains all the raw data from the experiments. Each ZIP archive includes the raw data corresponding to each figure, provided in either Prism or Excel format. The code used for the survival analysis is also included.
# Figure_1 Data.zip
This folder contains annotated sequence files and alignment data supporting Figure 1A and Figure 1B, which illustrate the genomic context, promoter structure, and sequence conservation of the Paillotin (IM18) peptide across dipteran species.
## File Descriptions
1. Fig1a_Paillotin_promoter_region.gb
- Genbank format file containing annotation of the Pai-RA/CG10332-RA and Pai-RB promoter regions.
- Highlights immune-related transcription factor binding motifs:
- GGRDNNHHBS (Relish-like)
- GGGHNNNDVH (Dif/Dorsal-like)
- Software: Can be opened with Geneious, SnapGene, or any GenBank-compatible viewer.
2. Fig1a_Paillotin_promoter_region.geneious
- Native Geneious project file containing the full annotation track, motif prediction, and aligned promoter sequences.
- This file requires Geneious software (commercial). For open alternatives, use the .gb file above.
3. Fig1a_subfile_IM18 region promoter.svg
- Visualization output (e.g. from Geneious) showing binding site arrangement within the promoter.
- Software: View/edit with Inkscape (free), Adobe Illustrator, or web browser.
4. Fig1b_Paillotin_alignment_May1-2025_v1.02
- FASTA or CLUSTAL format file with aligned Paillotin peptide sequences from >20 dipteran species.
-Open with Jalview, Geneious, or any alignment viewer supporting FASTA/NEXUS formats.
# Figure_2 Data.zip
This directory contains the qPCR data related to Figure 2 of the manuscript, focusing on the expression of Paillotin and CG10332 genes in Drosophila. The data were obtained using quantitative PCR (qPCR), and include both the raw Ct values and the normalized expression values.
## File Description
- Contains the raw and processed qPCR data for Paillotin and **CG10332 **expression upon E. coli and M. luteus bacterial infection.
1. CG10332-E.coli.csv
2. CG10332-M.luteus.csv
- Sheets include:
- Raw Ct: technical replicates for each condition
- Normalized: ΔCt and ΔΔCt values with fold-change
- Variables:
- Gene: target gene name
- Sample: genotype or treatment
- Ct: cycle threshold value
- Replicate: biological replicate ID
-Formula: Relative expression was calculated :
Expression ratio=(E ref)^Ct ref/(E target)^Ct target
where Rpl32 served as the reference gene.
For all runs the amplification efficiency (E) was assumed to be 2.00 unless stated otherwise.
3. Paillotin-E.coli.csv
4. Paillotin-M.luteus.csv
- Similar structure to the CG10332 file: raw Ct values, internal control (rpl32), and calculated expression values.
5. Figure 2-qpcr results.prism
- GraphPad Prism project containing bar graphs and statistical analyses of qPCR results for Figure 2. Includes grouped bar charts with error bars and statistical test results.
- Note: Requires GraphPad Prism software (licensed); for users without Prism, data is fully accessible in .csv files.
# Figure_3 Data.zip
This folder contains the raw data, processed spectra, and amino acid sequence alignments related to Figure 3 of the manuscript, which characterizes the Paillotin peptide using MALDI-TOF mass spectrometry.
## Folder Description
1. TIC MALDI-TOF.csv
This file contains Total-Ion-Current (TIC) MALDI-TOF mass-spectrometry data from Drosophila melanogaster haemolymph collected 24 h after infection with Erwinia carotovora strain Ecc15. All spectra were interpolated onto a common 0.02667 Da grid covering 4400-4800 Da and then normalised by each sample’s TIC.
-File layout
(Column letters assume Excel begins at A)
- A WT‑Reflectron m/z – raw, non‑uniform mass‑to‑charge axis for the WT spectrum recorded in reflectron mode.
- B WT‑intensity – raw ion counts corresponding to column A.
- C WT‑Reflectron m/z‑Interpolated – the common m/z grid in 0.02667 Da steps used for all spectra.
- D WT‑intensity‑Interpolated – WT intensities resampled onto the grid in column C.
- E 370‑intensity‑Interpolated – intensities for mutant 370 on the same grid.
- F SFHI‑intensity‑Interpolated – intensities for mutant SFHI on the same grid.
- G 41‑intensity‑Interpolated – intensities for line 41 on the same grid.
- H WT‑Total‑Ion‑Current (TIC) – single‑cell constant (row 1 only) storing the WT spectrum’s total ion count; blank below.
- I WT normalized intensity – WT intensities divided by the WT TIC (column H) and add a scaling factor 100000.
- J 370 TIC – constant TIC for mutant 370 (row 1 only).
- K 370 normalized intensity – 370 intensities divided by column J and add a scaling factor 100000.
- L SFHI TIC – constant TIC for mutant SFHI (row 1 only).
- M SFHI normalized intensity – SFHI intensities divided by column L and add a scaling factor 100000.
- N 41 TIC – constant TIC for line 41 (row 1 only).
- O 41 normalized intensity – line 41 intensities divided by column N and add a scaling factor 100000.
- Missing-data convention
NA: not applicable– the field has no logical value for that record
2. Figure 3-MALDI TOF.pzf
GraphPad Prism project file containing the normalized spectra and visualization shown in Figure 3(b), comparing the signal intensity of the Paillotin peptide (m/z ≈ 4626) in wild-type (w^1118) and three different mutant lines (Δ41, Δ370, ΔSFHI).
3. Pai amino acid sequence/
A folder containing amino acid sequences of Paillotin from wild-type (Dmel R6) and mutants (Δ41, Δ370, ΔSFHI), used to generate Figure 3(a).
- Software: Can be opened with Geneious or SnapGene.
###Data Processing Protocol
Data were processed as summarized below:
1. Raw Data Conversion:
MALDI-TOF output files were initially exported as .txt format.
2. Pre-processing:
- Files were imported into Origin for reformatting (space parsing, filtering m/z: 4400–4800).
- A new m/z axis was generated using a fixed step size (0.02667), with 15,000 points.
3. Interpolation & Alignment:
- Datasets with different m/z axes were interpolated using Origin (Analysis > Mathematics > Interpolate/Extrapolate).
- Intensities were aligned to the unified m/z axis.
4. Normalization:
- Data were normalized across samples using TIC-based normalization.
- Final datasets were exported to Excel for Prism import.
5. Visualization:
- Final spectra were plotted in GraphPad Prism.
- Figure 3(b) shows absence of the Paillotin peak in Δ41, Δ370, and ΔSFHI mutants relative to wild-type.
# Figure_4 Data.zip
This folder contains the qPCR data and visualization related to Figure 4 of the manuscript, analyzing the transcriptional response of DptA and Drosomycin (Drs) in different Drosophila genotypes upon bacterial infections.
## Folder Description
1. Dpt A-Ecc15.csv
- Contains the raw and processed qPCR data for Diptericin A expression upon Ecc15 bacterial infection.
- Timepoints: 6 hours post-infection (hpi) and 12 hpi, alongside unchallenged controls (UC).
- Includes Ct values for DptA and reference gene (RpL32), and normalized ΔCt and ΔΔCt values with fold-change.
- Variables:
- Sample: genotype or treatment
- Ct: cycle threshold value
- Replicate: biological replicate ID
2. Drs-M.luteus.csv
- Contains the raw and processed qPCR data for Drosomycin (Drs) expression following Micrococcus luteus bacterial infection.
- Timepoints: 8 hpi and 24 hpi, plus UC.
- Includes Ct values for Drs and reference gene (RpL32), and normalized ΔCt and ΔΔCt values with fold-change.
- Variables: the same as Dpt A-Ecc15.csv.
3. Figure 4.pzf
- GraphPad Prism project file used to generate the bar charts shown in Figure 4 (a–b).
- Subfigure (a): DptA expression (Imd pathway, Gram-negative bacteria).
- Subfigure (b): Drs expression (Toll pathway, Gram-positive bacteria).
# Figure_5 Data.zip
This folder contains data used to generate Figure 5, including survival records and CFU counts from Drosophila infection experiments.
## File Descriptions
1.P.burhodogranariea survival.prism
2.B.bassiana survival.pzfx
3.E.faecalis survival.prism
These Prism files contain daily survival records of different fly genotypes after infection with P. burhodogranariea, B. bassiana, and E. faecalis.
- 1 = dead, 0 = alive
4.CFU of P.bruhog.csv
This Excel file contains colony-forming unit (CFU) counts after infection with P. burhodogranariea.
- Variables: genotypes, replicates, dilution factors, fly counts, and calculated CFU values.
- The calculation formula: =[Count] * [DilutionFactor] * [[Total originally]] / ( [[uL plated]] * [[Total flies]] )
5.CFU of P.bruhog.prism
Graphs and statistics generated from the .xls CFU data.
# Figure_6 Data.zip
This folder contains raw data, statistical models, and visualization files for the analysis of survival phenotypes in flies after infection with Providencia burhodogranariea.
## File Descriptions
1. Pai+GroupB_cnv_P.burhodog.csv
- Excel file with original data used for survival analysis.
- Variables:
- Time2: time to death or censoring (in days)
- Censored: status (0 = censored, 1 = dead)
- Genotype, Sex, Experiment: categorical variables
- IM18(Paillotin) and GroupB: expression levels or binary genotype indicators
- Missing-data convention
- n/a: not applicable– the field has no logical value for that record
2. GroupB_Pai_CoxPH-12-12-2024.R
- R script that performs:
- Kaplan–Meier survival analysis with stratification by genotype
- Cox proportional hazards models (both fixed-effect and mixed-effect via coxme)
- Tests for interaction/synergy between IM18 and GroupB
- Export of data formatted for GraphPad Prism
3. Pai+GroupB_P.burhodog_survival.prism
- GraphPad Prism file containing survival records for different genotypes.
- Survival data is binary-coded:
- 1 = dead
- 0 = alive
# Figure_7 Data.zip
This file contains the survival data used in Figure 7, evaluating whether re-expression of Paillotin (IM18) can rescue susceptibility in IM18-deficient or immune-deficient mutant backgrounds upon Providencia burhodogranariea infection.
## File Description
1.Rescue survival.prism
- GraphPad Prism file with Kaplan–Meier survival data for multiple genotypes.
- Experimental groups include:
- Pai Δ41, Pai Δ370 mutants
- Rescue lines: Pai overexpression in Paillotin mutant and Relish mutant backgrounds
- Controls: w^1118, Rel^E20 alone
- Flies were pricked with P. burhodogranariea (OD600 = 12), and survival was monitored daily.
- Data format:
- 1 = dead
- 0 = alive
# Figure_S1 Data.zip
Folder: Pai nucleic acid sequence contains FASTA-format DNA sequences of Paillotin (IM18) homologs from different strains. These sequences were used for comparative analyses and sequence alignments.
- Pai-41.fasta
Nucleotide sequence of Paillotin from strain 41.
- Pai-370.fasta
Nucleotide sequence of Paillotin from strain 370.
- Pai-Dmel_R6.fasta
Reference IM18 (Paillotin) gene sequence from Drosophila melanogaster, genome release R6.
- Pai-SHFI.fasta
Nucleotide sequence of Paillotin from strain SHFI.
- Software: Can be opened with Geneious or SnapGene.
# Figure_S2 Data.zip
Figure_S2 Data contains Prism and Excel files used for the validation of Paillotin (IM18) gene expression via qPCR. These data support Supplementary Figure S2 of the manuscript.
## File Description
1.Paillotin-Ecc15.csv
Excel spreadsheet contains:
- Raw Ct: technical replicates for each condition
- Normalized: ΔCt and ΔΔCt values with fold-change
- Variables:
- Gene: target gene name
- Sample: genotype or treatment
- Ct: cycle threshold value
- Replicate: biological replicate ID
2.Figure S2.prism
GraphPad Prism file containing bar plots and statistical comparisons of qPCR results between wild-type and Paillotin mutant flies under different conditions.
# Figure_S3 Data.zip
Figure_S3 Data contains one Prism file showing survival curve of wt and Paillotin mutants after clean injury.
# Figure_S4 Data.zip
Figure_S4 Data contains eight Prism files with survival curve of wt and Paillotin mutants after different pathogen infections corresponding to figure 4 in the main text.
# Figure_S5 Data.zip
Figure_S5 Data contains a Prism file including time-course OD600 measurements and growth curve analyses of bacteria cultured with or without antimicrobial peptides, including Paillotin (IM18) and Cecropin A.
# Figure_S6 Data.zip
Figure_S6 Data contains all supporting data and scripts used to generate Figure S6A–C. The experiments test whether the loss of Paillotin (IM18) and GroupB-related immune factors result in increased susceptibility to various pathogens, and whether they act synergistically.
## File Descriptions
1. FigureS6A.prism
GraphPad Prism file showing survival of different fly genotypes following infection with P. rustigianii (OD = 2).
2. FigureS6B.prism
GraphPad Prism file containing survival data of different fly genotypes after infection with P. vermicola (OD = 0.1).
3. FigureS6C.prism
GraphPad Prism file containing survival data of different fly genotypes after infection with E. coli (OD = 200).
4. GroupB_Pai_CoxPH-12-12-2024.R
R script used to:
-Import survival data from Excel
-Convert data to GraphPad-compatible format
-Fit Cox proportional hazards and mixed-effect models
-Test for interaction/synergy between IM18 and GroupB
-Generate publication-quality Kaplan–Meier plots as PNG files
5. Pai+GroupB_cnv_P.rustigianii OD=2.csv
Raw survival data from P. rustigianii infection.
- Variables:
- Time2: time to death or censoring (in days)
- Censored: status (0 = censored, 1 = dead)
- Genotype, Sex, Experiment: categorical variables
- IM18(Paillotin) and GroupB: expression levels or binary genotype indicators
- Missing-data convention
- n/a: not applicable– the field has no logical value for that record
6. Pai+GroupB_cnv_P.vermicola OD=0.1.csv
Raw survival data from P. vermicola infection.
- Variables:
- Time2: time to death or censoring (in days)
- Censored: status (0 = censored, 1 = dead)
- Genotype, Sex, Experiment: categorical variables
- IM18(Paillotin) and GroupB: expression levels or binary genotype indicators
- Missing-data convention
- n/a: not applicable– the field has no logical value for that record
# Figure_S7 Data.zip
Figure_S7 Data contains one Prism file showing survival curve of Paillotin-overexpressed flies after bacteria infection.
# Figure_S8 Data.zip
Figure_S8 Data contains three Prism files showing survival curves of Paillotin mutants after different types of toxin infection corresponding Figure_S8 in the main text.
# Figure_S9 Data.zip
Figure_S9 Data contains the melanization rate and microbiota counting between wt and Paillotin mutants.
## File Descriptions
1. Figure S9A-Melanization.csv
Excel spreadsheet containing raw and summarized melanization scoring data for Drosophila infected with P. burhodogranariea.
2. Figure S9A-Melanization.pzfx
GraphPad Prism file used to generate bar graphs from melanization scores.
3. Figure S9B-Microbiota.csv
Excel file showing quantification of culturable gut microbiota of flies with different genotypes.
Variables include: CFU counts, fly genotype, dilution factor, fly numbers, and average CFU per fly. Formula is included in the sheet.
4. Figure S9B-Microbiota.prism
GraphPad Prism project containing microbiota CFU plots.
# Table_S1 Data.zip
This file summarizes natural genetic variation at the Paillotin (IM18) locus across multiple wild Drosophila melanogaster lines from the Drosophila Population Genomics Project Phase 3 (DPGP3).
## File Descriptions
1.Table S1 DPGP3_summary.csv
Contains the full population-level dataset, pairing detailed location metadata with fine-scale INDEL counts across the Paillotin gene for 30 DPGP3 populations.
2.Table S1 DPGP3_summary-1.csv
A streamlined population summary that retains overall mutation statistics and adds sub-continent aggregates for rapid regional comparisons.
3.Table S1 DPGP3_summary-2.csv
Collapses the same data to six continent-level rows, reporting total mutations, sample sizes and derived frequencies for broad global analyses.