Phase variation in Bacteroides fragilis governs susceptibility to a microvirus and drives its evolution

Abstract

The interaction and co-evolution between human gut bacteria and their phages shape the dynamic gut microbiome, exerting a significant impact on human health. However, the underlying mechanisms are largely unexplored. In particular, a bacteria-phage interaction model of the Bacteroidota phylum and the Microviridae phages is lacking, limiting our understanding of their ecological roles in human gut. In this study, we isolated a Bacteroidota-infecting Microviridae phage ϕHBP1 from human feces. Infection of its host Bacteroides fragilis with ϕHBP1 drives multiple genomic structural variations, which are correlated with host resistance to ϕHBP1. In turn, our phage evolution assay in B. fragilis H1 obtained ϕHBP1 mutants that carry mutations within the capsid and pilot proteins and can reinfect the resistant bacterial population. Together, our findings provide novel insights into an antagonistic co-evolution mechanism between gut phage and bacteria, and hold important implications for diversifying phages through evolution to target resistant bacteria in phage therapy.

Keywords

Microvirus / Bacteroidota / Bacteria-phage interaction / Phage evolution

Data availability statement

Raw sequencing for bacteria have been deposited under the NCBI BioProject with accession number PRJNA1258586. Genome of phage ϕHBP1 can be accessed in NCBI database via PV598014. Genome of B. fragilis H1 can be accessed in NCBI database via CP186030. Supplementary data associated with this article can be found online.

CRediT authorship contribution statement

Pan Huang: Writing - review & editing, Writing - original draft, Visualization, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Meiqi Du: Writing - review & editing, Writing - original draft, Visualization, Validation, Investigation, Data curation. Yanqiu Liu: Writing - review & editing, Visualization, Methodology, Investigation. Zhenhao Han: Writing - review & editing, Investigation. Qian Wan: Writing - review & editing, Investigation. Fuming Liang: Writing - review & editing, Investigation. Wenyuan Han: Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wenyuan Han reports financial support was provided by Huazhong Agricultural University. Wenyuan Han reports a relationship with Hubei Hongshan Laboratory that includes: board membership. Given his/her/their role as, Wenyuan Han had no involvement in the peer review of this article and had no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to another journal editor If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key Research and Development program of China 2022YFA0912200, Fundamental Research Funds for Central Universities 2662024SKPY003, Foundation of Hubei Hongshan Laboratory 2021hszd022, Hubei Special Project for Science Development 2024CSA060.

We extend our sincere thanks to all members of our laboratory for their valuable participation, insightful ideas, and suggestions, which greatly contributed to the study’s design and direction.

References

[1] K. Hou Z.X. Wu X.Y. Chen J.Q. Wang D. Zhang C. Xiao D. Zhu J.B. Koya L. Wei J. Li Z.S. Chen, Microbiota in health and diseases, Signal Transduct. Target Ther. 7 (2022) 135.

[2] J.C. Clemente, L.K. Ursell, L.W. Parfrey, R. Knight, The impact of the gut microbiota on human health: an integrative view, Cell 148 (2012) 1258-1270.

[3] E.Z. Gomaa, Human gut microbiota/microbiome in health and diseases: a review, Antonie Van Leeuwenhoek 113 (2020) 2019-2040.

[4] Z. Cao N. Sugimura E. Burgermeister M.P. Ebert T. Zuo P. Lan, The gut virome: a new microbiome component in health and disease, EBioMedicine 81 (2022) 104113.

[5] A. Chevallereau, B.J. Pons, S. van Houte, E.R. Westra, Interactions between bacterial and phage communities in natural environments, Nat. Rev. Microbiol. 20 (2022) 49-62.

[6] A.N. Shkoporov, C. Hill, Bacteriophages of the Human gut: the "known unknown" of the microbiome, Cell Host Microbe 25 (2019) 195-209.

[7] A.J. Hryckowian, B.D. Merrill, N.T. Porter, W. Van Treuren, E.J. Nelson, R.A. Gar- lena, D.A. Russell, E.C. Martens, J.L. Sonnenburg, Bacteroides thetaiotaomicron-in- fecting bacteriophage isolates inform sequence-based host range predictions, Cell Host Microbe 28 (2020) 371-379.e375.

[8] H. Zafar, M.H. Saier Jr., Gut Bacteroides species in health and disease, Gut Microbes 13 (2021) 1-20.

[9] H.M. Wexler, Bacteroides: the good, the bad, and the nitty-gritty, Clin. Microbiol. Rev. 20 (2007) 593-621.

[10] T. Martin, H. Aziz, Bacteroides fragilis: a case study of bacteremia and septic arthri- tis, Clin. Lab. Sci. 22 (2009) 131-135.

[11] N. Yutin, S. Benler, S.A. Shmakov, Y.I. Wolf, I. Tolstoy, M. Rayko, D. Antipov, P.A. Pevzner, E.V. Koonin, Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic fea- tures, Nat. Commun. 12 (2021) 1044.

[12] E. Guerin, A. Shkoporov, S.R. Stockdale, A.G. Clooney, F.J. Ryan, T.D.S. Sutton, L. A. Draper, E. Gonzalez-Tortuero, R.P. Ross, C. Hill, Biology and taxonomy of crAss-like bacteriophages, the most abundant virus in the Human gut, Cell Host Microbe 24 (2018) 6 653-664.e65.

[13] A.N. Shkoporov, E.V. Khokhlova, C.B. Fitzgerald, S.R. Stockdale, L.A. Draper, R.P. Ross, C. Hill, ΦCrAss 001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis, Nat. Commun. 9 (2018) 4781.

[14] N.T. Porter, A.J. Hryckowian, B.D. Merrill, J.J. Fuentes, J.O. Gardner, R.W.P. Glowacki, S. Singh, R.D. Crawford, E.S. Snitkin, J.L. Sonnenburg, E. C. Martens, Phase-variable capsular polysaccharides and lipoproteins mod- ify bacteriophage susceptibility in Bacteroides thetaiotaomicron, Nat. Microbiol. 5 (2020) 1170-1181.

[15] A.N. Shkoporov, E.V. Khokhlova, N. Stephens, C. Hueston, S. Seymour, A.J. Hryck- owian, D. Scholz, R.P. Ross, C. Hill, Long-term persistence of crAss-like phage crAss001 is associated with phase variation in Bacteroides intestinalis, BMC Biol. 19 (2021) 163.

[16] C.M. Krinos, M.J. Coyne, K.G. Weinacht, A.O. Tzianabos, D.L. Kasper, L.E. Com- stock, Extensive surface diversity of a commensal microorganism by multiple DNA inversions, Nature 414 (2001) 555-558.

[17] T. Kuwahara, A. Yamashita, H. Hirakawa, H. Nakayama, H. Toh, N. Okada, Kuhara, M. Hattori, T. Hayashi, Y. Ohnishi, Genomic analysis of Bacteroides frag- ilis reveals extensive DNA inversions regulating cell surface adaptation, Proc. Natl. Acad. Sci. 101 (2004) 14919-14924.

[18] A.M. Cerdeño-Tárraga, S. Patrick, L.C. Crossman, G. Blakely, V. Abratt, N. Lennard, I. Poxton, B. Duerden, B. Harris, M.A. Quail, A. Barron, L. Clark, C. Corton, J. Doggett, M.T. Holden, N. Larke, A. Line, A. Lord, H. Norbertczak, D. Ormond, C. Price, E. Rabbinowitsch, J. Woodward, B. Barrell, J. Parkhill, Extensive DNA in- versions in the B. fragilis genome control variable gene expression, Science 307 (2005) 1463-1465.

[19] E. Guerin, A.N. Shkoporov, S.R. Stockdale, J.C. Comas, E.V. Khokhlova, A.G. Clooney, K.M. Daly, L.A. Draper, N. Stephens, D. Scholz, R.P. Ross, C. Hill, Isolation and characterisation of ΦcrAss002, a crAss-like phage from the human gut that infects Bacteroides xylanisolvens, Microbiome 9 (2021) 89.

[20] E.M. Bean, N.M. Morella, N. Dey, Complete genome sequences of two Bacteroides uniformis bacteriophages, Microbiol. Resour. Announc. 11 (2022) e0061022.

[21] D.E. Campbell, L.K. Ly, J.M. Ridlon, A. Hsiao, R.J. Whitaker, P.H. Degnan, Infection with Bacteroides phage BV01 alters the host transcriptome and bile acid metabolism in a common Human gut microbe, Cell Rep. 32 (2020) 108142.

[22] A. Cortés-Martín, R. Denise, E. Guerin, S.R. Stockdale, L.A. Draper, R.P. Ross, A.N. Shkoporov, C. Hill, Isolation and characterization of a novel lytic Parabac- teroides distasonis bacteriophage 𝜑 PDS1 from the human gut, Gut Microbes 16 (2024) 2298254.

[23] J. Shen, J. Zhang, L. Mo, Y. Li, Y. Li, C. Li, X. Kuang, Z. Tao, Z. Qu, L. Wu, J. Chen, S. Liu, L. Zeng, Z. He, Z. Chen, Y. Deng, T. Zhang, B. Li, L. Dai, Y. Ma, Large-scale phage cultivation for commensal human gut bacteria, Cell Host Microbe 31 (2023) 665-677.e667.

[24] M.A. Tariq, F. Newberry, R. Haagmans, C. Booth, T. Wileman, L. Hoyles, M. R.J. Clokie, J. Ebdon, S.R. Carding, Genome characterization of a novel wastew- ater Bacteroides fragilis bacteriophage (vB_BfrS_23) and its host GB124, Front. Mi- crobiol. 11 (2020) 583378.

[25] P.C. Kirchberger, H. Ochman, Microviruses: a world beyond phiX174, Annu. Rev. Virol. 10 (2023) 99-118.

[26] T.A. Redgwell, J. Thorsen, M.A. Petit, L. Deng, G. Vestergaard, J. Russel, B. Chawes, K. Bønnelykke, H. Bisgaard, D.S. Nielsen, S. Sørensen, J. Stokholm, S.A. Shah, Prophages in the infant gut are pervasively induced and may modulate the func- tionality of their hosts, NPJ Biofilms Microbiomes 11 (2025) 46.

[27] M. Yehya, M. Hamze, H. Mallat, F. Dabbousi, Prevalence and antibiotic susceptibility of Bacteroides fragilis group isolated from stool samples in North Lebanon, Braz. J. Microbiol. 44 (2013) 807-812.

[28] R. Araujo, M. Muniesa, J. Méndez, A. Puig, N. Queralt, F. Lucena, J. Jofre, Optimisa- tion and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis, J. Virol. Methods 93 (2001) 127-136.

[29] D. Arndt, J.R. Grant, A. Marcu, T. Sajed, A. Pon, Y. Liang, D.S. Wishart, PHASTER: a better, faster version of the PHAST phage search tool, Nucleic Acids Res. 44 (2016) W16-W21.

[30] A. Bankevich, S. Nurk, D. Antipov, A.A. Gurevich, M. Dvorkin, A.S. Kulikov, V. M. Lesin, S.I. Nikolenko, S. Pham, A.D. Prjibelski, A.V. Pyshkin, A.V. Sirotkin, N. Vyahhi, G. Tesler, M.A. Alekseyev, P.A. Pevzner, SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing, J. Comput. Biol. 19 (2012) 455-477.

[31] T. Seemann, Prokka: rapid prokaryotic genome annotation, Bioinformatics 30 (2014) 2068-2069.

[32] J.R. Grant, E. Enns, E. Marinier, A. Mandal, E.K. Herman, C.Y. Chen, M. Graham, G. Van Domselaar, P. Stothard, Proksee: in-depth characterization and visualization of bacterial genomes, Nucleic Acids Res. 51 (2023) W484-w492.

[33] W. Li A. Godzik, Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences, Bioinformatics 22 (2006) 1658-1659.

[34] S. Capella-Gutiérrez, J.M. Silla-Martínez, T. Gabaldón, trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses, Bioinformatics 25 (2009) 1972-1973.

[35] L.-T. Nguyen, H.A. Schmidt, A. von Haeseler, B.Q. Minh, IQ-TREE: a fast and effec- tive stochastic algorithm for estimating maximum-likelihood phylogenies, Mol. Biol. Evol. 32 (2014) 268-274.

[36] J.P. Meier-Kolthoff, M. Göker, VICTOR: genome-based phylogeny and classification of prokaryotic viruses, Bioinformatics 33 (2017) 3396-3404.

[37] S. Nayfach, A.P. Camargo, F. Schulz, E. Eloe-Fadrosh, S. Roux, N.C. Kyrpides, CheckV assesses the quality and completeness of metagenome-assembled viral genomes, Nat. Biotechnol. 39 (2021) 578-585.

[38] S. Chen, Y. Zhou, Y. Chen, J. Gu, fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics 34 (2018) i884-i890.

[39] R.R. Wick, L.M. Judd, C.L. Gorrie, K.E. Holt, Unicycler: resolving bacterial genome assemblies from short and long sequencing reads, PLoS Comput. Biol. 13 (2017) e1005595.

[40] G. Marçais, A.L. Delcher, A.M. Phillippy, R. Coston, S.L. Salzberg, A. Zimin, MUM- mer4: a fast and versatile genome alignment system, PLoS Comput. Biol. 14 (2018) e1005944.

[41] A. Luque, S. Benler, D.Y. Lee, C. Brown, S. White, The missing tailed phages: predic- tion of small capsid candidates, Microorganisms 8 (2020).

[42] P.C. Kirchberger, Z.A. Martinez, H. Ochman, Organizing the Global diversity of mi- croviruses, mBio 13 (2022) e0058822.

[43] A.N. Shkoporov, A.G. Clooney, T.D.S. Sutton, F.J. Ryan, K.M. Daly, J.A. Nolan, S.A. McDonnell, E.V. Khokhlova, L.A. Draper, A. Forde, E. Guerin, V. Velayudhan, R.P. Ross, C. Hill, The Human gut virome is highly diverse, stable, and individual specific, Cell Host Microbe 26 (2019) 527-541.e525.

[44] A. Quaiser, A. Dufresne, F. Ballaud, S. Roux, Y. Zivanovic, J. Colombet, T. Sime-N- gando, A.J. Francez, Diversity and comparative genomics of Microviridae in Sphag- num- dominated peatlands, Front. Microbiol. 6 (2015) 375.

[45] M. Krupovic, P. Forterre, Microviridae goes temperate: microvirus-related proviruses reside in the genomes of bacteroidetes, PLoS One 6 (2011) e19893.

[46] L. Fernández-García, J. Kirigo, D. Huelgas-Méndez, M.J. Benedik, M. Tomás, R. Gar- cía-Contreras, T.K. Wood, Phages produce persisters, Microb. Biotechnol. 17 (2024) e14543.

[47] T. Kuwahara, A. Yamashita, H. Hirakawa, H. Nakayama, H. Toh, N. Okada, S. Kuhara, M. Hattori, T. Hayashi, Y. Ohnishi, Genomic analysis of Bacteroides frag- ilis reveals extensive DNA inversions regulating cell surface adaptation, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 14919-14924.

[48] S. Carasso, R. Keshet-David, J. Zhang, H. Hajjo, D. Kadosh-Kariti, T. Gefen, N. Geva-Zatorsky, Bacteriophage-driven DNA inversions shape bacterial function- ality and long-term co-existence in Bacteroides fragilis, Gut Microbes 17 (2025) 2501492.

[49] D. O’Sullivan, D.P. Twomey, A. Coffey, C. Hill, G.F. Fitzgerald, R.P. Ross, Novel type I restriction specificities through domain shuffling of HsdS subunits in lactococcus lactis, Mol. Microbiol. 36 (2000) 866-875.

[50] A. Anjum, K.J. Brathwaite, J. Aidley, P.L. Connerton, N.J. Cummings, J. Parkhill, I. Connerton, C.D. Bayliss, Phase variation of a Type IIG restriction-modification en- zyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168, Nucleic Acids Res. 44 (2016) 4581-4594.

[51] M.H. Foley, D.W. Cockburn, N.M. Koropatkin, The Sus operon: a model system for starch uptake by the human gut bacteroidetes, Cell Mol. Life Sci. 73 (2016) 2603-2617.

[52] R.M. Pollet, M.H. Foley, S.S. Kumar, A. Elmore, N.T. Jabara, S. Venkatesh, G. V. Pereira, E.C. Martens, N.M. Koropatkin, Multiple TonB homologs are impor- tant for carbohydrate utilization by Bacteroides thetaiotaomicron, J. Bacteriol. 205 (2023) e00218-e00223.

[53] D. Trzilova, R. Tamayo, Site-specific recombination - how simple DNA inversions produce complex phenotypic heterogeneity in bacterial populations, Trends Genet. 37 (2021) 59-72.

[54] I.R. Henderson, P. Owen, J.P. Nataro, Molecular switches-the ON and OFF of bac- terial phase variation, Mol. Microbiol. 33 (1999) 919-932.

[55] E.D. Sonnenburg, J.L. Sonnenburg, J.K. Manchester, E.E. Hansen, H.C. Chiang, J. I. Gordon, A hybrid two-component system protein of a prominent human gut sym- biont couples glycan sensing in vivo to carbohydrate metabolism, Proc. Natl. Acad. Sci. 103 (2006) 8834-8839.

[56] R. Gao T. Wu A.M. Stock, A conserved inhibitory interdomain interaction regulates DNA-binding activities of hybrid two-component systems in Bacteroides, mBio 15 (2024) e01220-e01224.

[57] J. Romeyer Dherbey, L. Parab, J. Gallie, F. Bertels, Stepwise evolution of E. coli C and PhiX174 reveals unexpected lipopolysaccharide (LPS) diversity, Mol. Biol. Evol. 40 (2023).

[58] A.M.S. Correa, C. Howard-Varona, S.R. Coy, A. Buchan, M.B. Sullivan, J.S. Weitz, Revisiting the rules of life for viruses of microorganisms, Nat. Rev. Microbiol. 19 (2021) 501-513.

[59] A.N. Shkoporov, C.J. Turkington, C. Hill, Mutualistic interplay between bacterio- phages and bacteria in the human gut, Nat. Rev. Microbiol. 20 (2022) 737-749.

[60] L. De Sordi, M. Lourenço, L. Debarbieux, The battle within: interactions of bacte- riophages and bacteria in the gastrointestinal tract, Cell Host Microbe 25 (2019) 210-218.

[61] Q. Zhong, B. Liao, J. Liu, W. Shen, J. Wang, L. Wei, Y. Ma, P.T. Dong, B. Bor, J.S. McLean, Y. Chang, W. Shi, L. Cen, M. Wu, J. Liu, Y. Li, X. He, S. Le, Episymbi- otic saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence, Proc. Natl. Acad. Sci. U.S.A. 121 (2024) e2319790121.

[62] L. Yang, J. Wang, S. Lu, Y. Zhong, K. Xiong, X. Liu, B. Liu, X. Wang, P. Wang, S. Le, Temperature-dependent carrier state mediated by H-NS promotes the long-term co- existence of Y. pestis and a phage in soil, PLoS Pathog. 19 (2023) e1011470.

[63] M.R. Mahmud, S.K. Tamanna, S. Akter, L. Mazumder, S. Akter, M.R. Hasan, M. Acharjee, I.Z. Esti, M.S. Islam, M.M.R. Shihab, M. Nahian, R. Gulshan, S. Naser, A.M. Pirttilä, Role of bacteriophages in shaping gut microbial community, Gut Mi- crobes 16 (2024) 2390720.

[64] J.M. Atack, C. Guo, L. Yang, Y. Zhou, M.P. Jennings, DNA sequence repeats iden- tify numerous type I restriction-modification systems that are potential epigenetic regulators controlling phase-variable regulons; phasevarions, Faseb J. 34 (2020) 1038-1051.

[65] A. Anjum, K.J. Brathwaite, J. Aidley, P.L. Connerton, N.J. Cummings, J. Parkhill, I. Connerton, C.D. Bayliss, Phase variation of a Type IIG restriction-modification en- zyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168, Nucleic Acids Res. 44 (2016) 4581-4594.

[66] N. Ben-Assa, M.J. Coyne, A. Fomenkov, J. Livny, W.P. Robins, M. Muniesa, V. Carey, S. Carasso, T. Gefen, J. Jofre, R.J. Roberts, L.E. Comstock, N. Geva-Zatorsky, Anal- ysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis, Nucleic Acids Res. 48 (2020) 11040-11053.

[67] P. Joglekar, E.D. Sonnenburg, S.K. Higginbottom, K.A. Earle, C. Morland, S. Shapiro-Ward, D.N. Bolam, J.L. Sonnenburg, Genetic variation of the SusC/SusD homologs from a polysaccharide utilization locus underlies divergent fructan speci- ficities and functional adaptation in Bacteroides thetaiotaomicron strains, mSphere (2018) 3.

[68] J.E. Emerson, C.B. Reynolds, R.P. Fagan, H.A. Shaw, D. Goulding, N.F. Fairweather, A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein, Mol. Microbiol. 74 (2009) 541-556.

[69] R. Gao T. Wu A.M. Stock, A conserved inhibitory interdomain interaction regulates DNA-binding activities of hybrid two-component systems in Bacteroides, mBio 15 (2024) e0122024.

[70] L. Zheng, Y. Tan, Y. Hu, J. Shen, Z. Qu, X. Chen, C.L. Ho, E.L. Leung, W. Zhao, L. Dai, CRISPR/Cas-based genome editing for Human gut commensal Bacteroides species, ACS Synth. Biol. 11 (2022) 464-472.

[71] L. García-Bayona, L.E. Comstock, Streamlined genetic manipulation of diverse Bac- teroides and parabacteroides isolates from the Human gut microbiota, mBio 10 (2019).

[72] P.C. Kirchberger, Z.A. Martinez, L.J. Luker, H. Ochman, Defensive hypervariable regions confer superinfection exclusion in microviruses, Proc. Natl. Acad. Sci. U.S.A. 118 (2021).

[73] J.M. Borin, S. Avrani, J.E. Barrick, K.L. Petrie, J.R. Meyer, Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance, Proc. Natl. Acad. Sci. U.S.A. 118 (2021).

[74] J.M. Borin, J.J. Lee, K.R. Gerbino, J.R. Meyer, Comparison of bacterial suppression by phage cocktails, dual-receptor generalists, and coevolutionarily trained phages, Evol. Appl. 16 (2023) 152-162.

[75] D. Maura, E. Morello, L. du Merle, P. Bomme, C.Le Bouguénec, L. Debarbieux, In- testinal colonization by enteroaggregative Escherichia coli supports long-term bacte- riophage replication in mice, Environ. Microbiol. 14 (2012) 1844-1854.

[76] A. Cortés-Martín, C. Buttimer, J.L. Maier, C.A. Tobin, L.A. Draper, R.P. Ross, M. Kleiner, C. Hill, A.N. Shkoporov, Adaptations in gut bacteroidales facilitate stable co-existence with their lytic bacteriophages, bioRxiv (2024).

[77] M. Lourenço, L. Chaffringeon, Q. Lamy-Besnier, T. Pédron, P. Campagne, C. Eberl, M. Bérard, B. Stecher, L. Debarbieux, L. De Sordi, The spatial heterogeneity of the gut limits predation and fosters coexistence of bacteria and bacteriophages, Cell Host Microbe 28 (2020) 390-401.e395.