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[Preprint]. 2024 Jun 19:2024.06.19.599758.

doi: 10.1101/2024.06.19.599758.

Pathogenic Bacteroides fragilis strains can emerge from gut-resident commensals

Renee E Oles^{1

2}, Marvic Carrillo Terrazas¹, Luke R Loomis¹, Maxwell J Neal³, Mousumi Paulchakrabarti⁴, Simone Zuffa^{5

6}, Chia-Yun Hsu¹, Adriana Vasquez Ayala¹, Michael H Lee², Caitlin Tribelhorn², Pedro Belda-Ferre², MacKenzie Bryant², Jasmine Zemlin^{5

6}, Jocelyn Young⁷, Parambir Dulai^{8

9}, William J Sandborn^{8

10}, Mamata Sivagnanam⁷, Manuela Raffatellu^{2

10

11}, David Pride^{1

10

12

13}, Pieter C Dorrestein^{5

6}, Karsten Zengler^{2

3

10

14}, Biswa Choudhury⁴, Rob Knight^{2

10

15

16

17}, Hiutung Chu^{1

10

11}

Affiliations

¹ Department of Pathology, University of California, San Diego, La Jolla, CA.
² Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
³ Department of Bioengineering, University of California, San Diego, La Jolla, CA.
⁴ GlycoAnalytics Core, University of California San Diego, San Diego, CA.
⁵ Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla.
⁶ Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA.
⁷ Division of Gastroenterology, Hepatology and Nutrition, University of California, San Diego and Rady Children's Hospital, San Diego, CA.
⁸ Division of Gastroenterology, University of California, San Diego, La Jolla, CA.
⁹ Division of Gastroenterology, Northwestern University, Chicago, Illinois.
¹⁰ Center for Microbiome Innovation, University of California, San Diego, La Jolla, CA.
¹¹ Chiba University-UC San Diego Center for Mucosal Immunology, Allergy and Vaccines (cMAV), University of California, San Diego, La Jolla, CA.
¹² Center for Innovative Phage Applications and Therapeutics (IPATH), University of California, San Diego, La Jolla, CA.
¹³ Center of Advanced Laboratory Medicine (CALM), University of California, San Diego, La Jolla, CA.
¹⁴ Program in Materials Science and Engineering, University of California, San Diego, La Jolla, CA.
¹⁵ Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA.
¹⁶ Department of Computer Science & Engineering, University of California, San Diego, La Jolla, CA.
¹⁷ Halıcıoğlu Data Science Institute, University of California, San Diego, La Jolla, CA.

PMID: 38948766
PMCID: PMC11213024
DOI: 10.1101/2024.06.19.599758

Pathogenic Bacteroides fragilis strains can emerge from gut-resident commensals

Renee E Oles et al. bioRxiv. 2024.

[Preprint]. 2024 Jun 19:2024.06.19.599758.

doi: 10.1101/2024.06.19.599758.

Authors

Affiliations

¹ Department of Pathology, University of California, San Diego, La Jolla, CA.
² Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, University of California, San Diego, La Jolla, CA.
³ Department of Bioengineering, University of California, San Diego, La Jolla, CA.
⁴ GlycoAnalytics Core, University of California San Diego, San Diego, CA.
⁵ Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla.
⁶ Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA.
⁷ Division of Gastroenterology, Hepatology and Nutrition, University of California, San Diego and Rady Children's Hospital, San Diego, CA.
⁸ Division of Gastroenterology, University of California, San Diego, La Jolla, CA.
⁹ Division of Gastroenterology, Northwestern University, Chicago, Illinois.
¹⁰ Center for Microbiome Innovation, University of California, San Diego, La Jolla, CA.
¹¹ Chiba University-UC San Diego Center for Mucosal Immunology, Allergy and Vaccines (cMAV), University of California, San Diego, La Jolla, CA.
¹² Center for Innovative Phage Applications and Therapeutics (IPATH), University of California, San Diego, La Jolla, CA.
¹³ Center of Advanced Laboratory Medicine (CALM), University of California, San Diego, La Jolla, CA.
¹⁴ Program in Materials Science and Engineering, University of California, San Diego, La Jolla, CA.
¹⁵ Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA.
¹⁶ Department of Computer Science & Engineering, University of California, San Diego, La Jolla, CA.
¹⁷ Halıcıoğlu Data Science Institute, University of California, San Diego, La Jolla, CA.

PMID: 38948766
PMCID: PMC11213024
DOI: 10.1101/2024.06.19.599758

Abstract

Bacteroides fragilis is a prominent member of the human gut microbiota, playing crucial roles in maintaining gut homeostasis and host health. Although it primarily functions as a beneficial commensal, B. fragilis can become pathogenic. To determine the genetic basis of its duality, we conducted a comparative genomic analysis of 813 B. fragilis strains, representing both commensal and pathogenic origins. Our findings reveal that pathogenic strains emerge across diverse phylogenetic lineages, due in part to rapid gene exchange and the adaptability of the accessory genome. We identified 16 phylogenetic groups, differentiated by genes associated with capsule composition, interspecies competition, and host interactions. A microbial genome-wide association study identified 44 genes linked to extra-intestinal survival and pathogenicity. These findings reveal how genomic diversity within commensal species can lead to the emergence of pathogenic traits, broadening our understanding of microbial evolution in the gut.

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Conflict of interest statement

Conflict of Interest W.J.S’s current conflicts of interest are: Mirador Therapeutics (stock, employee, company officer, Ventyx Biosciences (stock, former employee), Prometheus Laboratory (board of directors), Shoreline Biosciences (stock, scientific advisory board), Forbion (consultant), Alimentiv (consultant). P.C.D. is an advisor and holds equity in Cybele, BileOmix, and Sirenas and a Scientific co-founder, and advisor and holds equity to Ometa, Enveda, and Arome with prior approval by UC-San Diego. P.C.D. also consulted for DSM animal health in 2023. R.K.’s current conflicts of interest are: Gencirq (stock and SAB member), DayTwo (consultant and SAB member), Cybele (stock and consultant), Biomesense (stock, consultant, SAB member), Micronoma (stock, SAB member, co-founder), and Biota (stock, co-founder).

Figures

**Figure 1.. Pangenomic analysis of *Bacteroides fragilis* strains reveals extensive genetic variation.**
A) A core genome alignment-derived phylogenetic tree of *B. fragilis* strains by maximum-likelihood annotated with continent of isolate origin (located as dots at the tips), specific phylogroup clusters indicated by the gray inner ring, genetic regions of interest (the six colored rings which signify Type VI secretion system (T6SS) GA1 and GA3, pBFO42_2 and pBFO67_1, plasmid; *ubb; bft, B. fragilis* toxin with the three isoforms as separate shades of green (n=813; p-value of PERMANOVA of anatomic site = 0.133; p-value of PERMANOVA of geography = 0.087) with the anatomic site of isolation coloring the outermost ring. B) The number of genes that are strain-specific (in less than 1% of isolates), accessory (between 1% and 99% of isolates), or core (in more than 99% of isolates) in the *B. fragilis* pangenome of 813 strains. C) A measure of core compared with accessory genome evolution through cumulative gene gain/loss events (accessory genome) versus cumulative phylogenetic branch distance (core genome) (Student’s t-test, p = 3.41e-70), colored by terminal (light blue) and internal branches (dark blue) using the Panstripe algorithm (Tonkin-Hill et al., 2023).

**Figure 2.. *B. fragilis* phylogroups have unique core-phylogroup genes**
A) *B. fragilis* phylogroups based on t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm of a MASH, k-mer based distance matrix of the whole genome sequence of all strains. Phylogroups labeled 1–16. The commonly used laboratory strains, NCTC 9343, YCH46, and 638R, are labeled within their respective phylogroups along with other *B. fragilis* strains used in functional assays described in this study: HCBf046, HCBf084, HCBf077, and HCBf104. T6SS GA3^–/BFT⁺ represents a cluster of strains that lack T6SS GA3 and are positive for the presence of *bft*. B) Proportion of genes that are core to a collection of phylogroups, between 1 and 15, divided into COG categories with proportion expressed by the alpha of the heatmap cells. C) A heatmap of gene prevalence in each phylogroup. A euclidean clustering algorithm was applied to the rows and columns, gene clusters that belong to capsular polysaccharide paths are labeled, as well as the T6SS GA3 gene cluster and the location of *bft* and BfUbb. D) Western blot analysis of PSA of *B. fragilis* strains representing six PSA operon structures. Rabbit anti-PSA antibody was raised against NCTC 9343, which representing PSA operon 1 (top left). PSA operon structure of *B. fragilis* strains of high-quality assemblies (n=262). Genes colored by COG category and annotated with a gene name, if available by Bakta annotation.

**Figure 3.. Capsule heterogeneity among *B. fragilis* strains contributes to variable host immune responses.**
A) Transmission electron microscopy of six representative *B. fragilis* strains: NCTC 9343, HCBf046, HCBf104, 638R, HCBf077, and HCBf084. Phylogroup and PSA operon (P#/PSA#) are as indicated. Scale bar, 200 nm. B) Capsule analysis where mole percentage of monosaccharides of six representative *B. fragilis* strains was determined by GC-MS. Rib, ribose; Rha, rhamnose; Fuc, fucose; Man, mannose; Gal, galactose; Glc, glucose; GalA, galacturonic acid; Hep, heptose; KDO, ketodeoxyoctonic acid; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine. C) Capsule analysis where fatty acid percentage of six representative *B. fragilis* strains was determined by GC-MS. D) O-antigen analysis of whole cell lysates from six representative *B. fragilis* strains stained using Pro-Q Emerald glycoprotein stain. E) Serum killing assay of six representative *B. fragilis* strains. 10⁶ CFU of *B. fragilis* were treated with 0% or 60% normal human serum (NHS) in 1x PBS for 180 minutes. Results show the percent survival of *B. fragilis* strains. Two-way ANOVA comparing the mean of *B. fragilis* strains survival at 0% and 60% NHS. Data are representative of 3 experiments.

**Figure 4.. Growth and metabolic profiles of *B. fragilis* strains**
A) Growth of *B. fragilis* strains over time and normalized to untreated wells, as indicated by OD₆₀₀. Data is displayed as a heatmap, with a representative growth curve for NCTC 9343 overlaid above. Strains are ordered according to descending area under the curve (AUC). Data represents the AUC ± SEM, with the mean across three individual experiments. AUC coloring by extra-intestinal (pink), commensal (blue), and unknown (grey). B) PCA score plot of predicted metabolic fluxes, colored by commensal (blue) versus extra-intestinal (pink) strains. C) Defined carbon and nitrogen sources colored by growth (blue) or no growth (white) predicted for any *B. fragilis* strain. D) Predicted versus true growth of strains in defined nitrogen and carbon sources. E) Heatmap of differentially abundant metabolites compared to a media control, metabolites with annotation are labeled, hierarchical clustering of metabolites by row and strains by columns. Benjamini-Hochberg adjusted p ≤ 0.05 and log2-fold change ≥ 0.25; n of metabolites = 278; n of strains = 93.

**Figure 5.. Extra-intestinal strains have genetic features associated with their isolation site**
A) Genes identified through gene presence/absence mGWAS as differentially present in commensals versus extra-intestinal strains (dark blue, commensal; pink, extra-intestinal strains; Holm’s adjusted p≤0.05). Y axis is proportion of commensal or extra-intestinal strains with that gene as well as the percent difference in prevalence in commensal versus extra-intestinal strains. Size represents the −log10 p-value and color of each gene cluster determined by location on a pangenome graph, n=510 which excludes all metagenome assembled genomes (MAGs). B) Graphical representation of genes in the context of the pangenome in eight gene clusters. Group 1, commensal associated; Groups 2–7, extra-intestinal associated. The black border indicates genes which are predicted to be acquired through horizontal gene transfer (HGT). The pink border is highlighting the gene encoding the *B. fragilis* toxin, *bft.*

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References

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This is a preprint.

Pathogenic Bacteroides fragilis strains can emerge from gut-resident commensals

Affiliations

Pathogenic Bacteroides fragilis strains can emerge from gut-resident commensals

Authors

Affiliations

Abstract

Conflict of interest statement

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References

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