Journal: PLOS Biology

Article Title: An interbacterial cysteine protease toxin inhibits cell growth by targeting type II DNA topoisomerases GyrB and ParE

doi: 10.1371/journal.pbio.3003208

Figure Lengend Snippet: (a) The enzymatic reaction mechanism of papain-like cysteine proteases (PLCPs). The catalytic triad of residues in PLCPs, composed of cysteine (Cys), histidine (His), and aspartate (Asp), facilitates peptide bond cleavage. Histidine and aspartate form a proton-withdrawing group that activates the cysteine residue, converting it into a nucleophile. The activated cysteine attacks the carbonyl carbon of the peptide bond, leading to hydrolysis and bond cleavage. (b) Scaled depiction of representative genomic loci from the indicated species ( Edwardsiella tarda , Escherichia coli , Myxococcales bacterium , Roseofilum reptotaenium , Citrobacter rodentium , Rhizobium sp . , Burkholderia sp . ) encoding predicted PLCP domain-containing proteins (sky blue) and putative cognate immunity proteins (yellow). Regions encoding N-terminal domains of T6SS-related proteins (PAAR, VgrG, Rhs) are shaded in gray. CDI-related proteins (CdiA, CdiB) are depicted in light brown. Black bars designate locations of bases encoding the catalytic triads of PLCPs. Cysteine residues targeted for mutagenesis in this study are depicted in red. (c) Sequence logo generated from alignments of PLCP domains associated with bacterial contact-dependent antagonism pathways (the locus tag numbers for genes encoding the sequences we used are provided in ). Sequences from previously characterized PLCPs (ComA pep and LahT pep ) and recently identified T6SS-associated PLCP toxins (EvpQ and TsaP) are shown below for reference. (d) Maximum likelihood phylogeny of PLCP effectors visualized as a radiating phylogenetic tree. Taxonomy is indicated by colored shadows, and delivery pathways are indicated by colored tips. The scale bar represents the average number of substitutions per site. The original tree file used to generate this figure is available as . See also .

Article Snippet: E. coli S17-1 λ pir donor strains carrying the mutant constructs and E. coli ATCC-11775 recipient strains to be mutated were grown overnight on LB plates containing appropriate antibiotics, then scraped together to create a 1:1 mixture of each donor-recipient pair that was spread on an LB agar plate and incubated at 37 °C for 6 h to facilitate plasmid transfer via conjugation.

Techniques: Residue, Mutagenesis, Sequencing, Generated

Journal: PLOS Biology

Article Title: An interbacterial cysteine protease toxin inhibits cell growth by targeting type II DNA topoisomerases GyrB and ParE

doi: 10.1371/journal.pbio.3003208

Figure Lengend Snippet: (a) Viable E. coli cells recovered from plating cultures carrying plasmids expressing the indicated proteins on inducing media. E. coli carrying plasmid-borne Cpi1 or an empty vector was included to validate suppression of Cpe1 toxicity. Data are shown as the mean ± SD; n = 6. (b) Representative micrographs of E. coli cells expressing empty vector, Cpe1 tox , Cpe1 tox C362A , or co-expressing Cpe1 tox and Cpi1. Frames were acquired 2 h after induction of protein expression. Scale bar, 2 μm. (c) Ribbon diagram representation of the X-ray crystal structure of Cpe1 tox (sky blue) in complex with Cpi1 (light gold). Residues of the catalytic triad of Cpe1 tox are indicated. (d) Structural alignments of Cpe1 tox with previously characterized PLCPs: ComA pep (PDB: 3K8U, light gray) and LahT pep (PDB: 6MPZ, dark gray). (e) Bacterial competition experiments were conducted on a solid agar over a 4-h period between the indicated donor and recipient strains. E. coli ATCC-11775 ∆ hns served as the donor strain, whereas E. coli MG1,655 was used as the recipient strain. The ∆ hns mutation in the donor strain was introduced to up-regulate T6SS activity, as described in previous studies [ – ]. Data are shown as the mean ± SD; n = 3. (f) Depiction of Cpe1 tox illustrating the Cpe1 tox –Cpi1 interaction at the exosite formed by hydrophobic residues. (g) Magnified view of the interface between Cpe1 tox and Cpi1, highlighting the interacting residues on Cpi1 positioned near the hydrophobic concave surface of Cpe1 tox . (h) Viable E. coli cells recovered from E. coli co-expressing wild-type Cpe1 tox and the indicated Cpi1 variants. Data are shown as the mean ± SD; n = 6. (i) In vitro pull-down assay of Cpe1 tox and Cpi1. Cpe1 tox proteins were pulled down by strep-tagged Cpi1 variants and immobilized on streptavidin resin. Bound proteins were separated by SDS–PAGE followed by Coomassie brilliant blue staining. The data shown in (a) , (e) , and (h) are from a representative experiment of at least three independent experiments. P values were calculated with Student t test to assess differences in viability among populations (a, h) , and to evaluate statistically significant differences in the competitive indices of each donor strain against the specified recipients (e) . The data underlying this figure are available in and . See also , , and – .

Article Snippet: E. coli S17-1 λ pir donor strains carrying the mutant constructs and E. coli ATCC-11775 recipient strains to be mutated were grown overnight on LB plates containing appropriate antibiotics, then scraped together to create a 1:1 mixture of each donor-recipient pair that was spread on an LB agar plate and incubated at 37 °C for 6 h to facilitate plasmid transfer via conjugation.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Activity Assay, In Vitro, Pull Down Assay, SDS Page, Staining

Journal: PLOS Biology

Article Title: An interbacterial cysteine protease toxin inhibits cell growth by targeting type II DNA topoisomerases GyrB and ParE

doi: 10.1371/journal.pbio.3003208

Figure Lengend Snippet: (a) Viable E. coli cells recovered from expressing Cpe1 tox with substitutions of indicated surface residues from the hydrophobic concave region. Data are shown as the mean ± SD; n = 6. The data shown are from a representative experiment of at least three independent experiments. P -values were calculated with Student t test to compare each population’s viability with that of cells expressing wild-type Cpe1 tox . The expression levels of wild-type and Cpe1 tox variants were assessed by immunoblotting analysis, using the cytosolic protein RpoB as a loading control (right panel). (b) Schematic workflow for identifying candidate substrates of Cpe1 tox through Bpa crosslinking followed by LC–MS/MS protein identification. Bacteria carrying the construct with a TAG stop codon at residues of interest were grown in medium supplemented with Bpa. After incubation, cells were exposed to long-wavelength UV to activate formation of covalent bonds between Bpa and the interacting residues. Cell lysates were incubated with Ni-NTA resin to enrich for Cpe1 tox -Bpa crosslinks for further analysis. (c) SDS–PAGE gel analysis of crosslinked protein complexes before and after UV 365nm treatment. Cpe1 tox C362A -protein complexes enriched by the Ni-NTA resin were boiled in SDS sample buffer and separated by SDS–PAGE, followed by immunoblotting ( α -His). (d) Essential proteins identified in the Cpe1 tox C362A interactome and their biological functions. The complete interactome dataset is available in . (e, f, h, i) In vitro Cpe1 tox cleavage assays. Purified Cpe1 tox (wild-type or catalytic mutant) were incubated with purified candidate substrates, i.e., GyrB (e) and ParE (f) , or their variants with single-glycine deletion at the predicted cleavage sites (h, i) , to assess hydrolysis by Cpe1 tox . Fragmented proteins are marked with red arrowheads. Rxn means reaction. (g) Double glycine motifs in the consensus regions of E. coli GyrB and ParE. Locations of consensus regions between GyrB and ParE containing double glycine motifs are indicated on the cartoon representation of the full-length proteins. The sequences of the consensus regions are shown below, with the positions of the double glycine motifs labeled. The GG motif (red) and the surrounding residues (blue) are highlighted. The data underlying this figure can be found in and . The raw proteome data can be found in https://doi.org/10.5281/zenodo.15361709 .

Article Snippet: E. coli S17-1 λ pir donor strains carrying the mutant constructs and E. coli ATCC-11775 recipient strains to be mutated were grown overnight on LB plates containing appropriate antibiotics, then scraped together to create a 1:1 mixture of each donor-recipient pair that was spread on an LB agar plate and incubated at 37 °C for 6 h to facilitate plasmid transfer via conjugation.

Techniques: Expressing, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy, Bacteria, Construct, Incubation, SDS Page, In Vitro, Purification, Mutagenesis, Labeling

Journal: PLOS Biology

Article Title: An interbacterial cysteine protease toxin inhibits cell growth by targeting type II DNA topoisomerases GyrB and ParE

doi: 10.1371/journal.pbio.3003208

Figure Lengend Snippet: (a–b) Ribbon diagrams of E. coli GyrB (a, top structure) and ParE (b, top structure) before and after cleavage by Cpe1 tox . Crystal structures of the 43-kDa ATPase domain of E. coli GyrB (PDB: 4PRV) and ParE (PDB: 1S16) were used. Residues of the consensus sequence (LHAGGKF) are shown in yellow. Structure related to the small fragments after Cpe1 tox cleavage are indicated as pink ribbons. Predicted structures after cleavage at the double glycine motif demonstrated disruption of the ATPase domain of both GyrB (a, bottom structure) and ParE (b, bottom structure). (c, d) Sequence logos evidencing conservation of the consensus sequence (LHAGGKF) of GyrB (c) and ParE (d) homologs. Cleavage at the double glycine motif is indicated by dashed lines and a scissors illustration. (e) Plasmid DNA (pET) was analyzed by high-resolution agarose gel electrophoresis following isolation from E. coli that had expressed Cpe1 tox or the catalytic mutant for 0.5 or 1 h. The raw image is available in . (f) E. coli cells that had expressed Cpe1 tox , Cpe1 tox C362A , or Cpe1 tox co-expressed with Cpi1 for three hours. Cpe1 tox , or the catalytic mutant, was stained with DAPI (DNA; blue) and analyzed by fluorescence microscopy. Phase-contrast (top), blue fluorescence (middle), and merged (bottom) images are presented. Scale bar = 2 μm. Full-size images are available in . (g) Working model of how Cpe1 intoxicates bacteria. During interbacterial competition, Cpe1 targets and cleaves the ATPase domains of GyrB and ParE in target cells. The cleavage leads to accumulations of unrelaxed supercoils and precatenanes, disrupts DNA segregation, and ultimately inhibits cell division and growth. In the Cpe1-producing cells or kin cells expressing the immunity protein Cpi1, Cpe1 toxicity is neutralized through direct interaction between Cpe1 and Cpi1.

Article Snippet: E. coli S17-1 λ pir donor strains carrying the mutant constructs and E. coli ATCC-11775 recipient strains to be mutated were grown overnight on LB plates containing appropriate antibiotics, then scraped together to create a 1:1 mixture of each donor-recipient pair that was spread on an LB agar plate and incubated at 37 °C for 6 h to facilitate plasmid transfer via conjugation.

Techniques: Sequencing, Disruption, Plasmid Preparation, Agarose Gel Electrophoresis, Isolation, Mutagenesis, Staining, Fluorescence, Microscopy, Bacteria, Expressing

Journal: Cell host & microbe

Article Title: Bacteroides fragilis promotes chemoresistance in colorectal cancer, and its elimination by phage VA7 restores chemosensitivity.

doi: 10.1016/j.chom.2025.05.004

Figure Lengend Snippet: Figure 5. SusD/RagB is essential for B. fragilis-mediated chemoresistance (A) susD expression in stool samples from cohorts 1 and 2, as analyzed by HUMAnN3 from metagenomic data. (B) Deletion of susD using pSIE-KOsusD-bfe1-ErmR. The vector map of pSIE-KOsusD-bfe1-ErmR. TetR, Tet repressor protein, is the essential component of aTC-induced counter-selection system. TetO, Tet operator. bfe1, counter-selection marker. ErmR, resistance-selection marker. aTC, anhydrotetracycline hy- drochloride. Screening for positive cointegrates with erythromycin resistance. AmpR, conferring ampicillin resistance in E. coli S17-λ pir. (C) PCR analysis of B. fragilis mutants. Bacteroides fragilis (BF)-wild type (WT) (ATCC43860), BF mutant, and pSIE-KOsusD-Bfe1-ErmR were used as the template for the PCR (upper). Analysis of SusD/RagB protein expression of BF-WT and BFΔSusD/RagB (lower). (D) BFΔSusD/RagB failed to induce Notch1 activation in HT29 and HCT116 cells, as evidenced by western blot. (E) Schematic diagram showing experimental design of the effect of BFΔSusD/RagB on the response of HT29 xenografts to FOLFOX. FOLFOX chemotherapy regimen: weekly cycles of 6 mg/kg OXA followed 2 h later by 50 mg/kg 5-FU and 90 mg/kg folinic acid. (F) Tumor growth curve (left). Tumor images of HT29 xenografts under different treatments (middle). Tumor weight under different treatments at the endpoint (n = 8/group) (right). (G) PCR and western blotting validation for overexpression of SusD/RagB in E. coli MG1655 (EC-SusD/RagB).

Article Snippet: Genetic manipulation of B. fragilis Plasmid was transferred by conjugation from the donor strain E. coli S17- λ pir into the recipient enterotoxigenic B. fragilis strain ATCC 43860 as previously described.55,56 Overnight culture of E. coli S17- λ pir donor strain was diluted 250-fold in LB medium supplemented with ampicillin (100 μg/mL), and B. fragilis recipients were diluted 250-fold in Brain Heart Infusion (BHI) medium.

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Mutagenesis, Activation Assay, Western Blot, Biomarker Discovery, Over Expression

Journal: Cell host & microbe

Article Title: Bacteroides fragilis promotes chemoresistance in colorectal cancer, and its elimination by phage VA7 restores chemosensitivity.

doi: 10.1016/j.chom.2025.05.004

Figure Lengend Snippet: Figure 6. B. fragilis elimination efficacy, gut colonization ability, and safety of phage VA7 (A) In vitro lysis of B. fragilis and E. coli by phage VA7. Phage VA7 selectively suppressed the growth of B. fragilis in broth over 20 h, whereas the growth of E. coli was completely unaffected. (B) Scanning electron microscopy imaging visualized the stage of phage VA7 infecting B. fragilis. Scale bars represent 1 μm. (C) Transmission electron microscopy imaging visualized the lytic activity of phage VA7 to B. fragilis. (1) B. fragilis without phage infection. (2) Phages are infecting B. fragilis as indicated by arrows. (3) The contents of the lysed B. fragilis (left) and multiple phage particles around lysed bacteria (right). Scale bars represent 100 or 200 nm. (D) Schematic diagram of the shorter-term phage treatment experiment design. (E) Spot test assay validated that viable phage VA7 can be isolated from B. fragilis + phage VA7 mice stool. Positive results are expressed by clear lysis zones, as indicated by black arrows (right plate). (F) Relative abundance of B. fragilis decreased significantly after the oral administration of phage VA7, as determined by qPCR. B. fragilis levels were normalized to the 16S rRNA gene and compared with that at baseline. (G) PCR results of mice colonic total DNA amplified by VA7-specific primer (155 bp), as visualized by DNA electrophoresis, indicating phage VA7 can colonize in mouse colon tissues. (H) Schematic diagram of the longer-term phage treatment experiment design in AOM/DSS-induced CRC mice. (I) Spot test assay validated that viable phage VA7 can be isolated from the stool of mice in VA7 and B. fragilis + VA7 groups. Positive results are expressed by clear lysis zones (black arrow) (n = 5).

Article Snippet: Genetic manipulation of B. fragilis Plasmid was transferred by conjugation from the donor strain E. coli S17- λ pir into the recipient enterotoxigenic B. fragilis strain ATCC 43860 as previously described.55,56 Overnight culture of E. coli S17- λ pir donor strain was diluted 250-fold in LB medium supplemented with ampicillin (100 μg/mL), and B. fragilis recipients were diluted 250-fold in Brain Heart Infusion (BHI) medium.

Techniques: In Vitro, Lysis, Electron Microscopy, Imaging, Transmission Assay, Activity Assay, Infection, Bacteria, Spot Test, Isolation, Amplification, Nucleic Acid Electrophoresis