T. pallidum infection inhibited brain organoid neurodevelopment. (A) Schematic view of the methods for generating brain organoid from iPSCs. (B) Representative bright field images of individual brain organoids treated with T. pallidum over time. (C) Changes of the transcription levels of genes related to the three germ layers following T. pallidum infection in the brain organoids. (D) Immunofluorescent assay of the effect of T. pallidum infection on the brain organoid. A nonparametric t-test was used to evaluate the statistical differences between the two groups. (*: P < 0.05, **: P < 0.01, ***: P < 0.001, NS: not significant).

scRNA-seq revealed that T. pallidum infection inhibited the differentiation of neural progenitor cell subcluster 1B in the brain organoid. (A) Schematic overview of the 10×Genomics scRNA-seq procedure and analysis. (B) tSNE plot of single cells identified a total of 19 clusters. (C) tSNE plot of single cell depicting the separation into the 13 cell populations. (D) Distinguishing the tSNE plot of cell types and proportion analysis of 13 cell populations within the two groups. (E) Expression profile of key genes related to NPC1 and NPC2 cell populations between the two groups. Powder blue: control group; Pink: T. pallidum group; Pale green: NPC1; Yellow: NPC2; Red indicates high gene expression; Green indicates low gene expression. (F) Volcano plot of differential genes (T. pallidum vs control) in the NPC1 cluster. (G) GO analysis of the upregulated genes (left) and downregulated genes (right) in the NPC1 cluster following T. pallidum infection. (H) UMAP plot of distinguished NPC1 subclusters (left) and proportion analysis of each subcluster within two groups (right). Chi-square test was used to compare the changes in the proportion of the five small subgroups among the different groups. (I) Specificity marker expression of NPC1 subcluster.

T. pallidum infection inhibited the differentiation of neurones in the brain organoid. (A) Volcano Plots of differentially expressed genes related to neurons between the two groups. (B) Representative GO terms of downregulated and upregulated enrichment genes related to the neurons between two groups. (C)Violin diagram of genes related to early neuronal development. (D) The effect of T. pallidum infection on the transcription level of genes related to the nervous system in the brain organoids. (E) The frozen section of brain organoids by MAP2 fluorescence staining. (F) Flow sorting of brain organoids and statistical analysis of the two groups using a nonparametric t-test. (G) Cell morphology and validation of CD71−/CD49b− neuron populations. A nonparametric t-test was used to evaluate the statistical differences between the two groups. (*: P < 0.05, **: P < 0.01, ***, P <0.001, NS: not significant).

scRNA-seq revealed that T. pallidum infection inhibited the differentiation of the hindbrain neurons in the brain organoid. (A) UMAP embedding plots comparing the distribution of the brain organoids within the two groups. (B) UMAP embedding of cell subset in each region of the neurons calibrated by marker genes. (C) Subcluster cell number as a percentage of the neuron cluster cell number. The Chi-square test was used to compare the differences in the percentages among different cell subsets. (D) Violin diagram of the relevant marker genes in each neuronal region. (E) Immunohistochemistry of the brain organoids validating the expression of hindbrain neuron markers, including HOXA5 and SHOX2. A nonparametric t-test was used to compare the differences between the two groups (*: P < 0.05, **: P < 0.01, NS: not significant).

scRNA-seq revealed the mechanism of T. pallidum affecting neurodevelopment in the brain organoids. (A) Left panel: temporal analysis of subNPC1B-hindbrain neuron by Monocle 2, with subNPC1B subgroup in blue and hindbrain neuron subgroup in magenta. Right: HES4 localises to subNPC1B and NEUROGD1 localises to the hindbrain neuron. (B) Heatmap of differentially expressed transcripts along pseudotime. Cells are ordered by cell type and pseudotime. (C) Bubble difference diagram of subNPC1B and hindbrain neuron marker genes before and after T. pallidum infection. (D) GO analysis of the total number of differential genes in the subNPC1B subgroup. (E) The differential expression of transcription factors between the T. pallidum and control group by SCENIC analysis. (F) Sankey diagram showing the correspondence between differential transcription factors and neural differentiation and development. (G)Extended analysis of TCF3. (H) KEGG enrichment pathway analysis of hindbrain neuron subgroup.