Journal: eBioMedicine

Article Title: Gut bacterial type III secretion systems aggravate colitis in mice and serve as biomarkers of Crohn’s disease

doi: 10.1016/j.ebiom.2024.105296

Figure Lengend Snippet: A. pulmonis mAT1 harbors a functional T3SS. (A) Phylogenetic tree of A. pulmonis mAT1 (red), compared to A. dolens LMG 26841 strain and 14 closest type strains automatically picked by the Type Strain Genome Server (TYGS). The phylogenetic tree was generated by TYGS with default settings. (B) 35 online-available Achromobacter genomes were re-annotated by NR and searched for genes of T3SS proteins that belong to each T3SS ortholog. The quantities of each T3SS ortholog were plotted in the heatmap. Achromobacter strains were classified into host-isolated and environmental-isolated groups. Achromobacter strains in each group were organized by phylogenic tree generated by GTDB-Tk and IQ-TREE2 software. Achromobacter strains that contain more than 10 T3SS orthologs annotated by NR are considered T3SS-harboring strains and labeled red. (C) Neighbor-joining phylogenetic tree T3SS ATPase SctN of representative T3SS-harboring bacteria strains. Bacteria names are color-coded to indicate designated T3SS families. (D) T3SS gene cluster of A. pulmonis mAT1 compared with that of B. bronchiseptica RB50. Genes are color-coded to indicate designated components of the T3SS apparatus. (E) The relative expression level of T3SS genes of A. pulmonis mAT1 grown in designated media, measured by qRT-PCR. All data are expressed as the mean ± SEM of at least three experimental repeats, each with two technical replicates. The Kruskal–Wallis test with Dunne’s multiple comparisons was performed and adjusted by the Benjamini-Hochberg method. For Dunn’s multiple comparisons test, the mean of each group was compared with the mean of the control group. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Immunoblotting of secreted AopD by designated strains. Each bacterial culture was centrifuged to yield the “bacteria” fraction and the “supernatant” fractions. The fractions were separated by SDS/PAGE and transferred to polyvinylidene fluoride membranes. Membranes were immunoblotted with designated antibodies. DnaK served as a non-secreting and loading control. The data is representative of three experimental repeats.

Article Snippet: Proteins in both fractions were separated via 12% SDS-PAGE and analyzed by immunoblotting with a rabbit anti-AopD polyclonal antibody and a rabbit anti-DnaK polyclonal antibody (CUSABIO; #CSB-PA633459HA01EGW).

Techniques: Functional Assay, Generated, Isolation, Software, Labeling, Bacteria, Expressing, Quantitative RT-PCR, Control, Western Blot, SDS Page