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. 2023 Sep 27;11(5):e0017623.
doi: 10.1128/spectrum.00176-23. Online ahead of print.

The impact of Parkinson's disease-associated gut microbiota on the transcriptome in Drosophila

Xin Liu #  1   2 , Meng Yang #  2 , Runzhou Liu  2 , Fan Zhou  2 , Haibing Zhu  1   3 , Xiaoyun Wang  1   2
Affiliations

The impact of Parkinson's disease-associated gut microbiota on the transcriptome in Drosophila

Xin Liu et al. Microbiol Spectr. .

Abstract

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people, and many studies have confirmed that the disorder of gut microbiota is involved in the pathophysiological process of PD. However, the molecular mechanism of gut microbiota in regulating the pathogenesis of PD is still lacking. In this study, to investigate the impact of PD-associated gut microbiota on host transcriptome, we established various PD models with fecal microbiota transplantation (FMT) in the model organism Drosophila followed by integrative data analysis of microbiome and transcriptome. We first constructed rotenone-induced PD models in Drosophila followed by FMT in different groups. Microbial analysis by 16S rDNA sequencing showed that gut microbiota from PD Drosophila could affect bacterial structure of normal Drosophila, and gut microbiota from normal Drosophila could affect bacterial structure of PD Drosophila. Transcriptome analysis revealed that PD-associated gut microbiota influenced expression patterns of genes enriched in neuroactive ligand-receptor interaction, lysosome, and diverse metabolic pathways. Importantly, to verify our findings, we transplanted Drosophila with fecal samples from clinical PD patients. Compared to the control, Drosophila transplanted with fecal samples from PD patients had reduced microbiota Acetobacter and Lactobacillus, and differentially expressed genes enriched in diverse metabolic pathways. In summary, our results reveal the influence of PD-associated gut microbiota on host gene expression, and this study can help better understand the link between gut microbiota and PD pathogenesis through gut-brain axis. IMPORTANCE Gut microbiota plays important roles in regulating host gene expression and physiology through complex mechanisms. Recently, it has been suggested that disorder of gut microbiota is involved in the pathophysiological process of Parkinson's disease (PD). However, the molecular mechanism of gut microbiota in regulating the pathogenesis of PD is still lacking. In this study, to investigate the impact of PD-associated gut microbiota on host transcriptome, we established various PD models with fecal microbiota transplantation in the model organism Drosophila followed by integrative data analysis of microbiome and transcriptome. We also verified our findings by transplanting Drosophila with fecal samples from clinical PD patients. Our results demonstrated that PD-associated gut microbiota can induce differentially expressed genes enriched in diverse metabolic pathways. This study can help better understand the link between gut microbiota and PD pathogenesis through gut-brain axis.

Keywords: Drosophila melanogaster; Parkinson’s disease; fecal microbiota transplantation; gut microbiota; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Phenotypic analysis of the control and rotenone-treated Drosophila and validation of the models. (A) Experimental design of this study to show different groups of models, including control Drosophila (CTRL), Parkinson’s Drosophila (PD), control Drosophila with FMT from Parkinson’s Drosophila (CWFP), control Drosophila without FMT from Parkinson’s Drosophila (CWOFP), Parkinson’s Drosophila with FMT from control Drosophila (PWFC), and Parkinson’s Drosophila without FMT from control Drosophila (PWOFC). (B) Survival curves of CTRL and PD flies within 7 days (n = 80), significant differences were determined by log-rank test (P < 0.0001). (C) Negative geotaxis assays to show locomotor deficits of flies exposed to rotenone. Three replicates (20 flies in each replicate) were included in each timepoint and average numbers of flies were used for statistical analysis. (D) Relative expression of PD-related marker genes in CTRL and PD flies. Flies (male to female as 1:1) were used in the survival and locomotion assay. Significant differences are determined by the two-way ANOVA followed by Sidak’s post-hoc test. *P < 0.05, ***P < 0.001.
Fig 2
Fig 2
FMT significantly changed the composition of gut microbiota in each group Drosophila. (A) Analysis of β-diversity of gut microbiota in Drosophila. PCoA was used to calculate the distance between samples, and the clusters between groups were tested with PERMANOVA. Each point represented a sample, n = 5. (B) According to the species annotation information of OTUs/ASVs, the number of tags sequences of each sample at each classification level was counted. The vertical axis represented the percentage of each sample in the sequence at each classification level. (C) Gut microbiota analysis of bacterial structure in six groups at the genus level. (D) Chao1 α-diversity index of grouped data (n = 5). (E) Simpson α-diversity index of grouped data (n = 5). (F) Species Venn diagrams of six groups at the genus level. (G) The phenotypic distribution of each group analyzed by BugBase. (H) Relative abundance of pathogenic microbiota (n = 5). Control Drosophila (CTRL) and Parkinson’s Drosophila (PD) are flies before cross-colonization experiments (day 7). Control Drosophila with FMT from Parkinson’s Drosophila (CWFP), control Drosophila without FMT from Parkinson’s Drosophila (CWOFP), Parkinson’s Drosophila with FMT from control Drosophila (PWFC), and Parkinson’s Drosophila without FMT from control Drosophila (PWOFC) are flies after cross-colonization experiments (day 14). Significant differences are determined by the Wilcoxon test. *P < 0.05, **P < 0.01.
Fig 3
Fig 3
RNA sequencing analysis of Drosophila samples before and after FMT experiments. (A) Pearson correlation heatmap of RNA-seq data from CTRL and PD groups. (B) PCA of RNA-seq data showed the separation of six groups, n = 3. (C) Venn diagram showed the numbers of DETs between four pairs of comparisons. (D) Volcano plot showed DETs between CTRL group and PD group. (E) Volcano plot showed DETs between CWFP group and CWOFP group. (F) Volcano plot showed DETs between PWFC group and PWOFC group. (G) Volcano plot showed DETs between PWOFC group and CWOFP group. (H) Enrichment analysis of KEGG pathway between CTRL group and PD group. (I) Enrichment analysis of KEGG pathway between CWFP group and CWOFP group. (J) Enrichment analysis of KEGG pathway between PWFC group and PWOFC group. (K) Enrichment analysis of KEGG pathway between PWOFC group and CWOFP group. The x-axis represented enrichment factors and the y-axis represented different pathways of biological processes.
Fig 4
Fig 4
Correlations analysis of functional pathways influenced by PD-associated gut microbiota and RT-qPCR validation of genes. (A) Functional prediction heatmap with PICRUSt2. Vertical axis indicated functional classification, horizontal axis indicated samples, and the color indicated the richness of the function. (B) Indicator species analysis at genus level of six groups. (C) Correlation analysis of the four gut microbes with DETs, which are representative genes from pathways of “neuroactive ligand-receptor interaction” and “lysosome” in Fig. 3. (D) Relative expression of transcripts encoding neuroactive ligand receptor interactions in CTRL group and PD group. (E) Relative expression of transcripts encoding neuroactive ligand-receptor interaction in CWFP group and CWOFP group. (F) Relative expression of transcripts encoding neuroactive ligand-receptor interactions in PWFC group and PWOFC group. Significant differences were determined by the unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5
Fig 5
Microbial structure and diversity in Drosophila colonized with fecal samples from clinical patients. (A) Schematics diagram showing experimental design of this study. (B) Analysis of β-diversity of gut microbiota in FMT-CTRL and FMT-PD groups from conventional Drosophila. PCoA was used to calculate the distance between samples, and the clusters between groups were tested with PERMANOVA. Each point represents a sample, n = 7 or 8. (C) Welch’s t-test showing differences in species abundance for conventional Drosophila and germ-free Drosophila, *P < 0.05. (D) Venn diagram indicating the numbers of species in two groups. (E) Indicator value (Indval) of species in each group at the genus level. (F) Functional prediction heatmap with PICRUSt2. (G) Analysis of β-diversity of gut microbiota in GF-CTRL and GF-PD groups from germ-free Drosophila. PCoA was used to calculate the distance between each sample. Each point represents a sample, n = 8. (H) Indicator value (Indval) of species in each group at the genus level. (I) Venn diagram indicating the numbers of species in two groups.
Fig 6
Fig 6
Analysis of Drosophila transcriptome transplanted with PD-associated gut microbiota from clinical patients. (A) Pearson correlation heatmap of RNA-seq data from FMT-PD group and FMT-CTRL group, n = 3. (B) PCA of RNA-seq data from FMT-PD group and FMT-CTRL group, n = 3. (C) Volcano plot showing differentially expressed genes of Drosophila transplanted with PD-associated gut microbiota. (D) Enrichment analysis of the KEGG pathway of differentially expressed genes. (E) Relative expression level of genes associated with tyrosine metabolism in RT-qPCR. (F) Relative expression level of genes associated with retinol metabolism in RT-qPCR. Significant differences are determined by the unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
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