Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a Schematic representation of the B. fragilis NCTC9343 (hereafter referred to as NCTC9343) T6SS locus. Predicted gene products are shown above the indicated genes. Core T6SS structural components are shaded in gray; five hcp variants are colored in red. b Experimental design for antibiotic treatment and competitive colonization in C57BL/6 J mice. Numbers indicate days before (−) and after (+) strains gavage. c In vivo competition assays between killer strains (wild-type NCTC9343, T6SS-deficient mutants Δ tssC or Δ hcp1 – hcp5 ) and a Bte1-sensitive prey strain (Δ tssC ΔV1-ErmR) in antibiotic-treated C57BL/6 J mice (5 mice per group). Fecal samples collected at specified time points were homogenized in PBS, diluted, and plated on selective BHI agar (200 μg/mL gentamycin + 15 μg/mL erythromycin) for c.f.u. counting. The black dashed line indicates the limit of detection (L.O.D.). Data are presented as the arithmetic mean ± SEM. Unpaired two-tailed Student’s t-tests were used for comparing means between two groups. Source data are provided as a Source Data file. d In vitro co-culture assays between the indicated killer and prey strains. Wild-type NCTC9343 (WT) and its isogenic deletion mutants (Δ tssC or Δ hcp1 – hcp5 ) serve as killers. Bte1 sensitive mutant (NCTC9343 ΔV1-CamR) serves as prey. Prey survival was determined by serial dilution and plating on chloramphenicol-supplemented BHI agar. e Immunoblot analysis of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT) and its isogenic deletion mutants (Δ tssC or Δ hcp1 – hcp5 ). DnaK serves as a cytoplasmic loading control. f In vitro co-culture assays using NCTC9343 (WT), Δ tssC , Δ hcp2 , Δ hcp3 , Δ hcp2 - hcp3 ( Δ hcp23) or the indicated plasmid-based complementary strains (Δ hcp2::phcp2 , Δ hcp3::phcp3 , Δ hcp2 - hcp3::phcp2-hcp3 ) as killers and Bte1 sensitive mutant (NCTC9343 ΔV1-CamR) as prey. Prey survival was determined by serial dilution and plating on chloramphenicol-supplemented BHI agar. g Immunoblot detection of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT), its isogenic deletion mutants (Δ tssC , Δ hcp2 , Δ hcp3 or Δ hcp2 - hcp3 ) and the indicated plasmid-based complementary strains (Δ hcp2::phcp2 , Δ hcp3::phcp3 or Δ hcp2 - hcp3::phcp2-hcp3 ). For ( d – g ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: In Vivo, Two Tailed Test, In Vitro, Co-Culture Assay, Mutagenesis, Serial Dilution, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a In vivo pull-down analysis to detect the interactions of Hcp1, Hcp2, Hcp3 and Bte1 in NCTC9343, hcp3 deletion strain or hcp2 deletion strain. Whole cell lysates were used as input for in vivo pull-down. The 6 × His-tagged Bte1 was enriched using Ni-affinity resin. The coprecipitated proteins (output) were detected by western immunoblots with antibodies specific to the indicated proteins. b In vitro pull-down analysis to detect the interactions of Hcp2, Hcp3 and Bte1. The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. c In vitro pull-down analysis to detect the interactions of Hcp1 and Bte1. The strep-tagged Hcp1 was enriched using Strep-affinity resin. No interaction between Hcp1 and Bte1 was detected. d Top view 2D class averages of the in vitro purified Hcp2-Hcp3 heterohexamers obtained from cryoSPARC. The unique α-helix in Hcp2 (red dashed box) was used to determine subunit stoichiometry. Both 3:3 and 4:2 (Hcp2:Hcp3) assemblies were observed in vitro. e Sequence alignment of Hcp2 NCTC9343 and Hcp3 NCTC9343 . The key amino-acid residues used to distinguish Hcp2 and Hcp3 in the cryo-EM map are indicated by arrows. f Close-up of cryo-EM densities identifying key side chains distinguishing Hcp2 (Tyr55, Ile106) and Hcp3 (Ser53, Arg103), thereby confirming the presence of a 4:2 Hcp2-Hcp3 stoichiometry in the in vitro assembled heterohexamers. g Cryo-EM map of the Hcp2 4 -Hcp3 2 heterohexamer (top and side views), with Hcp2 shown in dark sea green and Hcp3 in violet. h Cartoon representation of the Hcp2 4 -Hcp3 2 heterohexamer, shown with C2 symmetry. Individual Hcp subunits and their corresponding chain IDs are labeled. i – k Molecular details of the three distinct subunit interfaces between Hcp2 and Hcp3 (red, blue, yellow boxes in h ), stabilized by hydrophobic and main-chain polar interactions. Interacting residues are shown in stick, and polar interactions are indicated by black dashed lines. For ( a – c ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: In Vivo, Western Blot, In Vitro, SDS Page, Staining, Purification, Sequencing, Cryo-EM Sample Prep, Labeling

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a Top (left) and side (right) views of the cryo-EM map of the Bte1-Hcp2 4 -Hcp3 2 complex. Proteins are color-coded: Bte1 (steel blue and cyan), Hcp2 (dark sea green), Hcp3 (violet). b Ribbon model of the Bte1-Hcp2 4 -Hcp3 2 ternary complex. The Hcp ring was modeled using the apo Hcp2 4 -Hcp3 2 structure to resolve low-density regions. Bte1 was built using its resolved N-terminal region and a ModelAngelo-built C-terminal helical domain. The composite Bte1-Hcp2-Hcp3 model was generated for illustrative purposes. c Crystal structure of Bte1 (gold) fitted into the Bte1-Hcp2 4 -Hcp3 2 cryo-EM density (gray). In this conformation, helix α3 sterically clashes with the Hcp2-Hcp3 complex, whereas helices α5 and α6 are positioned at the ring-ring stacking interface of the Hcp tube, thereby potentially interfering with tube assembly. d Structural comparison of crystal structure of Bte1 (gold) versus cryo-EM-resolved Bte1 (steel blue) and ModelAngelo-bulit Bte1 C-terminal helical domain (Cyan) reveals conformational rearrangements in α3, α5, and α6. e Top views 2D class averages of the two stoichiometries of the in vitro purified Bte1-Hcp2-Hcp3 complex obtained from cryoSPARC. Bte1 density and the unique α-helix in Hcp2 are boxed in blue and red, respectively. f The N-terminal region of Bte1 inserts into the central channel of the Hcp2-Hcp3 complex and interacts with a pair of adjacent Hcp2-Hcp3 subunits. Two interaction regions are highlighted by yellow and red boxes, respectively. g Electrostatic surface analysis of the Hcp2-Hcp3 and Bte1 interaction interface. The molecular surfaces are colored by electrostatic potential (red, negative; blue, positive). Key regions of electrostatic complementarity, which are critical for the binding interaction, are highlighted with black dashed boxes. The electrostatic potential scale ranges from − 6 kT/e (red) to + 6 kT/e (blue). h Close-up views of the Bte1-Hcp2 4 -Hcp3 2 interface. Two polar interaction regions are highlighted (yellow and red dashed boxes from f ). Black dashed lines indicate polar contacts. i In vitro pull-down analysis to detect the interactions of Bte1 with Hcp2-Hcp3 complex or its indicated mutants. The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. Mutation of Hcp2 N30A or Hcp3 R100A within the complex abolished Bte1 binding. j In vitro co-culture assays using wild-type NCTC9343 (WT), Δ hcp2 - hcp3 , or plasmid-based hcp2-hcp3 point mutant complementation strains as killers, with the Bte1-sensitive mutant (NCTC9343 ΔV1-CamR) as prey. Prey survival was assessed via 10-fold serial dilution and plating on chloramphenicol-supplemented BHI. k Immunoblot detection of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT), Δ hcp2-hcp3 , or plasmid-based hcp2-hcp3 point mutant complementation strains. DnaK serves as a cytoplasmic loading control. l Sequence alignment of Hcp2 and Hcp3 homologs from multiple Bacteroides strains indicates that critical Bte1-interacting residues (Asn30 in Hcp2 and Arg100 in Hcp3) are not conserved. For ( i – k ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: Cryo-EM Sample Prep, Generated, Comparison, In Vitro, Purification, Binding Assay, SDS Page, Staining, Mutagenesis, Co-Culture Assay, Plasmid Preparation, Serial Dilution, Western Blot, Expressing, Control, Sequencing

Journal: Nature Communications

Article Title: Effector conformational plasticity enables lineage-specific secretion via Hcp heterohexamers in gut symbionts

doi: 10.1038/s41467-026-69309-z

Figure Lengend Snippet: a Maximum-likelihood phylogenetic analysis of Hcp2-Hcp3 fused proteins from 38 B. fragilis strains, revealing 4 distinct subtypes. Strains encoding the three V1 effectors characterized in this study are highlighted in red. V1/V2-associated effectors are indicated next to each strain. Hcp2 and Hcp3 are tightly linked to V1, but not V2, effectors. Identified effectors are labeled by name; uncharacterized ones are noted as putative. Gradient bars reflect sequence identity to B. fragilis NCTC9343 Hcp2 and Hcp3. Bootstrap values (> 70%) are shown at nodes. b In vitro co-culture assays using NCTC9343 (WT), Δ tssC , Δ hcp2-hcp3 , or indicated plasmid-based complementation strains (Δ hcp23::phcp23 NCTC9343 , Δ hcp23::phcp23 GS086 and Δ hcp23::phcp23 HMW616 ) as killers, with Bte1-sensitive mutant (NCTC9343 ΔV1-CamR) as prey. Prey survival was quantified by 10-fold dilution plating on chloramphenicol-supplemented BHI. c Immunoblot detection of Bte1 and Hcp1 expression and secretion in wild-type NCTC9343 (WT), its isogenic deletion mutants (Δ tssC or Δ hcp2 - hcp3 ) and the indicated plasmid-based complementary strains (Δ hcp23::phcp23 NCTC9343 , Δ hcp23::phcp23 GS086 or Δ hcp23::phcp23 HMW616 ). d In vitro pull-down analysis to detect the interactions between Bte1 or Bfe1 and the Hcp2-Hcp3 complex from either NCTC9343 or GS086. The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. Each Hcp2-Hcp3 complex selectively bound a particular V1 region effector. e , f In vitro pull-down analysis to detect the interaction of Bte1 ( e ) or Bfe1 ( f ) with different Hcp2-Hcp3 combinations: Hcp2 NCTC9343 -Hcp3 NCTC9343 , Hcp2 GS086 -Hcp3 GS086 , Hcp2 NCTC9343 -Hcp3 GS086 , and Hcp2 GS086 -Hcp3 NCTC9343 . The strep-tagged Hcp2 was enriched using Strep-affinity resin, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining. Complexes containing Hcp3 NCTC9343 bound Bte1 but not Bfe1. In contrast, those containing Hcp3 GS086 bound Bfe1 but not Bte1. g Surface representation of the Bte1-bound Hcp2-Hcp3 complex colored by sequence conservation. The surface is colored by sequence identity from cyan (0% variable) to pink (100% conserved). The global sequence identity values derived from individual Hcp2 and Hcp3 homologs across different strains are indicated. For ( b – f ), representative results are shown from experiments that were conducted at least three times with consistent results. Source data are provided as a Source Data file.

Article Snippet: The contents of the gels were transferred to PVDF membranes (Millipore), blocked, and probed with primary antibodies α-Dnak (cytoplasmic control), α-Bte1 and α-Hcp1 (Rabbit α-Bte1 and α-Hcp1, this study, used at a dilution of 1:3000; Rabbit α-DnaK, Cusabio #CSB-PA633459HA01EGW, used at a dilution of 1:3000; Mouse α-HA, MBL #M180-3, used at a dilution of 1:2000), followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, MBL #458, diluted to 1:5000; goat anti-mouse, MBL #330, diluted to 1:5000).

Techniques: Labeling, Sequencing, In Vitro, Co-Culture Assay, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, SDS Page, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Evolutionary engineering a larger porin using a loop-to-hairpin mechanism

doi: 10.1101/2023.06.14.544993

Figure Lengend Snippet: (A, B) Western blots with the nitrocellulose membrane stained with Ponceau S and subsequently immunoblotted against various antibodies. (A) Western blot of different fractions of E. coli displaying the localization of the his-tagged recombinant proteins PorB WT and PorB 18 in the outer membrane. SurA, a periplasmic protein, indicates the localization of periplasmic proteins. Whole-cell fractions (WC), soluble (S), inner membrane and trapped periplasmic protein (IM/P), and outer membrane (OM) (B) Western blot displaying the susceptibility of proteins to proteinase K under different conditions. His-tagged PorB WT (blue asterisks) and PorB 18 (green asterisks) show resistance to proteinase K when not boiled but are partially susceptible when heat denatured. TolC (red asterisks) is used as a positive control as it shows a 5 kDa cleavage in the presence of proteinase K resulting in a 45 kDa cleavage product (red arrows) .

Article Snippet: The following antibodies were diluted as per manufacturer recommendation with TBST with 1% gelatin: THE Anti-His tag monoclonal mouse antibody (GenScript), anti- E. coli SurA polyclonal rabbit antibody (Cusabio), IRDye 800CW goat anti-mouse IgG secondary antibody (LI-COR Biosciences), IRDye 680RD donkey anti-rabbit IgG secondary antibody (LI-COR Biosciences). anti- E. coli TolC was purified from rabbit sera and diluted to 1:2500 with 1% gelatin in TBST.

Techniques: Western Blot, Membrane, Staining, Recombinant, Positive Control

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: Fig. 1: 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

Article Snippet: The proteins in the supernatant fraction were precipitated in 10% (v/v) trichloroacetic acid.26 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