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Bacteroides fragilis ubiquitin homologue drives intraspecies bacterial competition in the gut microbiome

Nature Microbiology volume 9, pages 70–84 (2024)Cite this article

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Abstract

Interbacterial antagonism and associated defensive strategies are both essential during bacterial competition. The human gut symbiont Bacteroides fragilis secretes a ubiquitin homologue (BfUbb) that is toxic to a subset of B. fragilis strains in vitro. In the present study, we demonstrate that BfUbb lyses certain B. fragilis strains by non-covalently binding and inactivating an essential peptidyl-prolyl isomerase (PPIase). BfUbb-sensitivity profiling of B. fragilis strains revealed a key tyrosine residue (Tyr119) in the PPIase and strains that encode a glutamic acid residue at Tyr119 are resistant to BfUbb. Crystal structural analysis and functional studies of BfUbb and the BfUbb–PPIase complex uncover a unique disulfide bond at the carboxy terminus of BfUbb to mediate the interaction with Tyr119 of the PPIase. In vitro coculture assays and mouse studies show that BfUbb confers a competitive advantage for encoding strains and this is further supported by human gut metagenome analyses. Our findings reveal a previously undescribed mechanism of bacterial intraspecies competition.

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Fig. 1: BfUbb exhibits potent bactericidal toxicity depending on the C-terminal disulfide bond.
Fig. 2: BfUbb functions in the periplasm causing elongation, swelling and lysis of sensitive strains.
Fig. 3: BfUbb targets a periplasmic PPIase to antagonize sensitive B. fragilis.
Fig. 4: Determination of BfUbb–PPIase binding specificity.
Fig. 5: BfUbb exerts bactericidal toxicity by impairing the prolyl isomerase and chaperone activities of targeted essential PPIase.
Fig. 6: BfUbb increases the competitiveness of its encoding strains in intraspecies competition.

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Data availability

The atomic coordinates and structure factors generated in the present study have been deposited in the PDB under the accession nos. 8HM1, 8HM2, 8HM3 and 8HM4. The whole-genome sequence of B. fragilis GS077 and GS084 was deposited into GenBank under accession nos. JAVFHL000000000 (for GS077) and CP133097 (for GS084). Source data are provided with this paper.

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Acknowledgements

We thank A. L. Goodman (Yale University) for technical support and J. Galan (Yale University), Y. Shi (Westlake University) and H. Long (Ocean University of China) for discussion and careful review of the manuscript. We thank the staff from BL02U1 and BL18U1 beamlines of the National Facility for Protein Science Shanghai at SSRF for assistance during data collection. We also thank Y. Guo, J. Qu, J. Zhu, Z. Li, S. Wang, X. Zhao, H. Yu, X. Li and G. Lin from the core facilities for life and environmental sciences, SKLMT of Shandong University for their assistance in laser-scanning confocal microscopy, LC–MS/MS and X-ray diffraction experiments. The present study was supported by the National Natural Science Foundation of China (grant no. 32122007 to X.G.), the National Key R&D Program of China (grant no. 2022YFA1304200 to X.G.), the Shandong Provincial Natural Science Foundation (grant nos. ZR2021JQ09, ZR2019ZD21 and 2020CXGC011305 to X.G.) and the Youth Interdisciplinary Innovative Research Group of Shandong University (grant no. 2020QNQT009 to X.G.).

Author information

Author notes

  1. These authors contributed equally: Kun Jiang, Weixun Li, Ming Tong, Jinghua Xu, Zhe Chen.

Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Kun Jiang, Weixun Li, Ming Tong, Jinghua Xu, Zhe Chen, Yan Yang, Yuanrong Zang, Xuyao Jiao, Chang Liu, Dalei Wu, Mingyu Wang, Shuang-Jiang Liu & Xiang Gao

  2. Department of Microbial Pathogenesis and Microbial Sciences Institute, Yale University School of Medicine, New Haven, CT, USA

    Bentley Lim

  3. Microbiome Research Center, Moon (Guangzhou) Biotech Co. Ltd., Guangzhou, China

    Xianzhi Jiang

  4. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Jiawei Wang

  5. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Shuang-Jiang Liu

  6. National Institute of Biological Sciences, Beijing, China

    Feng Shao

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Contributions

X.G. and K.J. conceived the project. X.G., K.J., W.X.L., M.T., J.H.X., Z.C., C.L., B.L. and J.W.W. provided the methodology. X.G., K.J., W.X.L., M.T., J.H.X., Z.C., Y.Y, Y.R.Z. and X.Y.J. did the investigations. K.J., W.X.L., M.T., J.H.X. and Z.C. validated the results. X.G., K.J., W.X.L., M.T., Z.C. and F.S. wrote the original draft of the manuscript. X.G., K.J., W.X.L., D.L.W, B.L., F.S. and S.J.L. reviewed and edited the manuscript. X.G. acquired funding. X.G., X.Z.J, M.Y.W. and S.J.L. provided the resources. X.G. supervised the project.

Corresponding author

Correspondence to Xiang Gao.

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Nature Microbiology thanks Han Remaut, Kevin Foster and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Screening of BfUbb sensitive B. fragilis strains.

a, Agar spot analysis is used to screen BfUbb-sensitive B. fragilis strains on a BfUbb-containing plate. 22 B. fragilis strains were spotted on a BHIS plate spread with 100 μg of BfUbb to evaluate their BfUbb sensitivity. The selected BfUbb-sensitive strains are listed on the right and underlined in red. b, Minimum inhibitory concentration (MIC) assays of BfUbb, tetracycline (tet), and chloramphenicol (cam) on B. fragilis GS077 and GS076. Strains were cultured in BHIS medium containing BfUbb, tet, or cam with the indicated gradient concentrations. Dotted lines represent the 50% concentrations of the control strains (CT). Data are expressed as the mean ± s.d. from three independent experiments. c, Western immunoblot analyses of BfUbb in the whole cell lysate (WCL) and culture supernatant (Sup) of strains of B. fragilis NCTC 9343 wildtype, ubb deleted mutant and ubb restored mutant. d, 5 µl culture supernatant of B. fragilis NCTC 9343 wildtype, ubb deleted mutant, and ubb restored mutant are spotted on the BHIS plates and then overlaid with a layer of agar containing the indicated strains. For a, c, and d, experiments were conducted at least three times with consistent results.

Source data

Extended Data Fig. 2 Morphology changes of B. fragilis GS077S or GS084I exposed to BfUbb.

a, b, Confocal microscopy images show the morphology of B. fragilis GS077S and GS084I exposed to BfUbb at concentrations of 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, and 10 μg/ml for 5 h. c, The linear correlation between BfUbb’s protein loading and band intensity was assessed through quantitative western blot analysis. The data are presented as the mean ± s.d. from at least three independent experiments, chosen to be representative of the overall trend. The true protein loading versus protein band intensity is depicted by the black curve, while the fitted linear relationship is represented by the dashed red line. The figure displays the specific mapping formula and coefficient of determination. d, Confocal microscopy images show the morphology of B. fragilis GS077S after being treated with the supernatant of B. fragilis NCTC 9343 for 4 h. e, Western immunoblot analysis of the amount of BfUbb secreted by ~4×107 B. fragilis NCTC 9343 (S1), the minimal amount of BfUbb needed to induce substantial elongation and growth defects in ~3.6×107B. fragilis GS077S (In), and the remaining BfUbb in the supernatant (S2). Data are representative of three independent experiments. f, Histograms showing the amount of BfUbb secreted by ~1×107 B. fragilis NCTC 9343 and the minimal amount of BfUbb needed to induce substantial elongation and growth defects in ~1×107B. fragilis GS077S. Data are expressed as the mean ± s.d. from three independent experiments. For b and d, Cellular membranes are stained with lipophilic dye FM 1-43 (green); scale bar, 5 μm; the images represent at least 10 images from three independent experiments.

Source data

Extended Data Fig. 3 Sequence alignment of PPIase proteins from B. fragilis NCTC 9343, GS077S, and GS084I.

Amino acid sequence alignment of the PPIase proteins from BfUbb-sensitive strain GS077S, BfUbb-insensitive strain GS084I, and BfUbb-encoding strain B. fragilis NCTC 9343.

Extended Data Fig. 4 PPIaseGS077 is the target of BfUbb.

a, b, Size-exclusion chromatography analyses of BfUbb in the presence of PPIaseGS077 (a) or PPIaseGS084 (b). Purified BfUbb was incubated with purified PPIaseGS077 or PPIaseGS084 and subsequently analysed using size-exclusion chromatography in a Superdex 75 increase column. Elution profiles along with SDS-PAGE analyses of the elution fractions are shown. This experiment was conducted at least three times with consistent results. c, d, f, The binding affinities of BfUbb (c and d) and BfUbbC70V (f) to PPIaseGS077 (c and f) or PPIaseGS084 (d) were measured by ITC. The N values were fixed to 1 during the data fitting for d and f. The Kd and ΔH values data are representative of 2-3 independent replicates. e, Confocal microscopy views of B. fragilis GS077S treated with BfUbb, BfUbbC70V, and BfUbbC76G after 5 hours, respectively. Cellular membranes are stained with lipophilic dye FM 1-43 (green); scale bar, 5 μm; the images represent at least 20 images from three independent experiments.

Source data

Extended Data Fig. 5 Tyr119 in PPIase is essential for interacting with BfUbb.

a, Confocal microscopy views of B. fragilis GS077S, GS077-PPIGS084(110-120), GS084I, and GS084-PPIGS077(110-120) treated with BfUbb after 5 hours. In strain GS077-PPIGS084(110-120), amino acids 110-120 of PPIaseGS077 were replaced with the corresponding amino acids 110-120 of PPIaseGS084. In strain GS084-PPIGS077(110-120), amino acids 110-120 of PPIaseGS084 were replaced with the corresponding amino acids 110-120 of PPIaseGS077. b, Confocal microscopy views of B. fragilis GS077S, GS077-PPIE118A/Y119E, GS084I, and GS084-PPIA118E/E119Y treated with BfUbb for 5 hours. c, Confocal microscopy views of B. fragilis GS077S, GS077-PPIE118A, GS077-PPIY119E, GS084I, GS084-PPIA118E, and GS084-PPIE119Y treated with BfUbb for 5 hours. For a, b, and c, cellular membranes are stained with lipophilic dye FM 1-43 (green); scale bars, 5 μm; the images represent at least 20 images from three independent experiments.

Extended Data Fig. 6 Crystal structures of the BfUbb–PPIaseGS077 complex and BfUbbC70V/C76G.

a, Structural alignment of the two copies of BfUbb–PPIaseGS077 complex in one asymmetric unit. The disulfide bond formed by Cys70 and Cys76 in BfUbb and the key interacting residue Y119 in PPIaseGS077 are shown as sticks. The black arrow indicates the angle of rotation around the central axis. b, c, The binding affinities of BfUbbD75G (b) and BfUbbN7A (c) to PPIaseGS077 were measured by ITC. The N values were fixed to 1 during the data fitting. The Kd and ΔH values data are representative of 2-3 independent replicates. d, Hydrophobic interactions between R8/I44/L68/I69/C70/C76 of BfUbb (light blue) and Y56/L55/L52 of PPIaseGS077 (green). e, f, B-factor diagram of BfUbb (e) and BfUbbC70V/C76G (f) represented by the B-factor putty program in PyMOL. The cartoon thickness and color reflect the relative Cα B-factors within the molecule. The disulfide bond between C70 and C76 in BfUbb is shown as a yellow stick.

Extended Data Fig. 7 The ratio of PPIase that needs to be targeted by BfUbb to see a cell growth defect.

a, The linear correlation between PPIase protein loading and band intensity was assessed through quantitative western blot analysis. The data are presented as the mean ± s.d. from at least three independent experiments. b, Confocal microscopy images of B. fragilis GS077S treated with BfUbb (0.15 μg/ml) after 3 h. c, Western immunoblot analysis of PPIase and BfUbb from ~8×107 cells of B. fragilis GS077S after incubation with BfUbb (0.15 μg/ml) for 3 h. Data are representative of at least three independent experiments. d, e, Histograms showing the amount (d) and concentration (e) of PPIase and BfUbb in ~1×107 cells of B. fragilis GS077S after they were treated with BfUbb (0.15 μg/ml) for 3 h. f, Confocal microscopy images of morphological changes in B. fragilis GS077ΔPPIaseGS077 harboring a plasmid expressing PPIaseGS077 in BHIS containing 0.1 mM rhamnose. The wild-type strain was used as the negative control. g, Western immunoblot analysis of the amount of PPIase in ~1×107 cells of B. fragilis GS077S and B. fragilis GS077ΔPPIaseGS077 harboring a plasmid expressing PPIaseGS077 in BHIS containing 0.1 mM rhamnose. Data are representative of three independent experiments. h, Histograms showing the relative concentration of PPIase in ~1×107 cells of B. fragilis GS077S and B. fragilis GS077ΔPPIaseGS077 harboring a plasmid expressing PPIaseGS077 in BHIS containing 0.1 mM rhamnose. The concentration of PPIase in B. fragilis GS077S is normalized as 100%. i, Co-immunoprecipitation analysis detecting the interaction of BfUbb with PPIase from B. fragilis GS077S when the majority of GS077S cells are exhibiting growth defects. j, Relative concentrations of BfUbb versus PPIase in Extended Data Fig. 7i. The concentration of PPIase is normalized as 100%. For b and f, Cellular membranes are stained with lipophilic dye FM 1-43 (green); scale bar, 5 μm; the images represent at least 10 images from three independent experiments. For d, e, h, and j, Data are expressed as the mean ± s.d. from three independent experiments.

Source data

Extended Data Fig. 8 Crystal structure of the PPIaseGS077.

a, Overall structure of PPIaseGS077. The two PPIaseGS077 molecules in one asymmetric unit are shown in cartoon models. b, c, Electron density maps showing the conformations of individual domains of the PPIaseGS077 in one asymmetric unit: chain A (b) and chain B (c). The 2Fo–Fc electron density maps of the chain A and chain B are contoured at 1.2 σ. d, e, PPIaseGS077 molecules in one asymmetric unit from the BfUbb–PPIaseGS077 complex are shown as cartoon models. The key residue Y119 is labeled and shown as a stick. f, g, PPIaseGS077 molecules in one asymmetric unit from the crystal structure PPIaseGS077 are shown as cartoon models. The key residue Y119 is highlighted in red. For a-g, NTD, N-terminal domain; CTD, C-terminal domain; P1 and P2, PPIase domain.

Extended Data Table 1 BfUbb-interacting proteins identified in GS077S
Full size table

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

List of bacterial strains used in the present study.

Supplementary Table 2

Data collection and refinement statistics.

Supplementary Table 3

The LC–MS/MS analysis results of BfUbb-interacting proteins in GS077S.

Supplementary Table 4

Identification and co-occurrence of BfUbb, BfUbb-targeting PPIase, BfUbb-encoding strains, PPIase and BfUbb-insensitive PPIase homologues in human gut metagenomes EMBL.

Supplementary Table 5

Identification and co-occurrence of BfUbb, BfUbb-targeting PPIase, BfUbb-encoding strains, PPIase and BfUbb-insensitive PPIase homologues in human gut metagenomes 3CGC.

Supplementary Table 6

List of plasmids used in the present study.

Supplementary Table 7

Nucleotide sequences of PPIases used in thie present study.

Supplementary Video 1

Time-lapse of B. fragilis GS077S showing the morphology changes over the course of 8 s after being treated with BfUbb for 5 h. Cellular membranes are stained with lipophilic dye FM 1-43 (green). Scale bar, 10 μm. Taken under ×63 oil-immersion objective from three independent experiments.

Source data

Source Data Fig. 1

Uncropped plate corresponding to Fig. 1g.

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Uncropped plates, gels and western blots corresponding to Fig. 3c–e,h.

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Statistical source data for Fig. 3g.

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Uncropped plates and gels corresponding to Fig. 4b–g,l,m,o.

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Uncropped plates corresponding to Fig. 5a–c.

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Statistical source data for Fig. 5f–i.

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Statistical source data for Fig. 6a–f,h–l.

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Uncropped western blots and gels corresponding to Extended Data Fig. 1c,d.

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Statistical source data for Extended Data Fig. 1b.

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Uncropped western blots corresponding to Extended Data Fig. 2c,e.

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Statistical source data Extended Data for Fig. 2c,f.

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Uncropped Coomassie Blue gels for Extended Data Fig. 4a,b.

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Statistical source data for Extended Data Fig. 4a,b.

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Uncropped western blots corresponding to Extended Data Fig. 7a,c,g,i.

Source Data Extended Data Fig. 7

Statistical source data for Extended Data Fig. 7a,d,e,h,j.

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Jiang, K., Li, W., Tong, M. et al. Bacteroides fragilis ubiquitin homologue drives intraspecies bacterial competition in the gut microbiome. Nat Microbiol 9, 70–84 (2024). https://doi.org/10.1038/s41564-023-01541-5

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