The basis of antigenic operon fragmentation in Bacteroidota and commensalism

Abstract

The causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism within the phylum Bacteroidota remain unclear (1, 2). Using the classical lipopolysaccharide/O-antigen 'rfb operon' in Enterobacteriaceae as a surface antigen model (5-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification (3), we characterized the architecture/conservancy of the entire rfb operon in Bacteroidota. Analyzing complete genomes, we discovered that most Bacteroidota have the rfb operon fragmented into non-random gene-singlets and/or doublets/triplets, termed 'minioperons'. To reflect global operon integrity, duplication, and fragmentation principles, we propose a five-category (infra/supernumerary) cataloguing system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in specific micro-niches. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes (4). DNA insertions overrepresenting DNA-exchange-avid species, impact functional metagenomics by inflating gene-based pathway inference and overestimating 'extra-species' abundance. Using bacteria from inflammatory gut-wall cavernous micro-tracts (CavFT) in Crohn's Disease (5), we illustrate that bacteria with supernumerary-fragmented operons cannot produce O-antigen, and that commensal/CavFT Bacteroidota stimulate macrophages with lower potency than Enterobacteriaceae, and do not induce peritonitis in mice. The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism offers potential for novel diagnostics and therapeutics.

Keywords: Alistipes; Bacteroides; Escherichia coli; O-antigen; Parabacteroides; Prevotella; Salmonella.

Figure 1.. The rfb operon in Enterobacteriaceae is contiguous, but in Bacteroidota it is often fragmented into ‘minioperon’.

A) Lipopolysaccharide (LPS) and O-antigen schematics. B) Classical rfb operon with 5 contiguous genes in E. coli K12 (rfbXABCD; XCADB; [rfb:5]). Horizontal lines represent the bacterial genome length, distribution of rfb genes/operons (shaded circles; darker = more genes) and rfb gene orientation (+, sense; -, antisense). nL: n of rfb clusters or gene singlet loci. Note orientations/duplications. C) Five categories of operon arrangement & ‘minioperon’ fragmentation in Bacteroidota. Supplementary Figure 1C illustrates in context rfb operons/fragmentation for Alistipes, Bacteroides, Parabacteroides, Prevotella, Paraprevotella, Barnesiella, Tannerella, Odoribacter and Porphyromonas.

Figure 2.. The occurrence, patterns and inversions of rfb minioperons in P. distasonis and B. thetaiotaomicron suggests mechanism of operon fragmentation in Bacteroidota.

A) Fragmentation of rfb operon in P. distasonis is in modern times supernumerary compared to ATCC8503 strain from USA/1933. B) Fragmentation has resulted in conserved singlet, doublet/triplet patterns in P. distasonis. C) Heatmap clustering of P. distasonis strains based on rfb minioperon shows distinct clades. Additional information is available in Supplementary Figure 2B. D) Unique rfbFGC->rfbA minioperon distancing pattern (downward red/black arrows and solid circles) in B. thetaiotaomicron is also present in novel CavFT strains of P. distasonis from gut wall lesions in Crohn’s disease. Notice the orientation sense and patterns. E) SDS-PAGE of LPS extract analysis of E. coli and P. distasonis CavFT-46 shows that P. distasonis is unable to produce O-antigen polysaccharides in diverse growth conditions (Fisher’s exact P=0.079).

Figure 3.. The rfb operon in Alistipes is contiguous and duplicated suggesting evolutionary benefit.

A) Alistipes has contiguous rfb operons with frequent duplication and less common incorporation of rfb minioperon duplets. B) Patterns of conserved minioperons in Alistipes differ from Parabacteroides. C) Schematics of gene-gene rfb distances measured within and between minioperons. D) Parabacteroides and Bacteroides demonstrate the greatest variance in number of rfb operon fragments and gene-gene distances. Alistipes and Enterobacteriaceae are similarly contiguous. Intergene distances for Parabacteroides, Bacteroides and Prevotella were greater than Enterobacteriaceae (0.54±0.84Mb, 0.49±0.86Mb, 0.34±0.32Mb, respectively, vs. 0.13±0.46Mb, P<0.001). Alistipes gene distribution is similar to Enterobacteriaceae (0.17±0.49Mb, P=0.79). E) Minioperon sequence homologies for Alistipes vs. Parabacteroides based on minioperon orientation between and within genera/species. Two tailed-T tests P<0.01 **, P<0.001 ***. F) Alignment and G) phylogeny based on rfb operon sequences. Note that Alistipes clusters are driven by the sense/antisense orientation of the operons.

Figure 4.. ‘Rfb-Operon Profiling’ indicates non-random selection and minioperon similarity between Parabacteroides and Bacteroides, namely B. thetaiotaomicron.

A) Gaps and insertions in rfb gene sequences designate different rfb-types using protocols described for rfbA-typing (3). Supplementary Figure 4 illustrates the rfb-typing of rfbC/D/F/G. B) Example of global rfb operon profiling system (GOPS) for P. distasonis. C) Density plots between random and real rfb operon profiles in P. distasonis. Observed types are statistically different from a random (uniform) distribution (**, *** for P<0.05 and P<0.01, respectively). D) Phylogenetics across Bacteroidota and Enterobacteriaceae based on rfb mini/operon sequences. Remarkably, several Bacteroides species, but namely B. thetaiotaomicron CLT5T119C52, cluster together with several Parabacteroides, especially P. distasonis, irrespective of minioperon considered (red squares; further details in Supplementary Figures 5).

Figure 5.. Fragmentation of nonessential operons is driven by insertions of ‘foreign DNA’, mainly Bacteroides.

A) Schematics of O-antigen (rfb), fimbriae (fim) and ribosomal (rrn) gene operons tested for DNA insertions. B) Example of P. distasonis inter-minioperon DNA fragment found in other genomes. C) % of DNA from one species common to the genome of 4–5 other species within the genus. D) Schematics showing source of ‘foreign DNA’ insertions into the rfbFGC->rfbA space separating the rfb genes in P. distasonis. E) ‘Pure’ P. distasonis DNA fragments intermixed within inter-minioperon ‘foreign DNA’ insertions. F) Genus sources and % of ‘foreign DNA’ fragmenting the rfb and fim minioperons in P. distasonis, as in Figure 5A. Bacteroides (B. thetaiotaomicron, B. fragilis, B. ovatus, B. vulgatus) are the main source of ‘foreign DNA bombardment’ (P<0.0001 vs. Alistipes and Escherichia). Additional information is available in Supplementary Table 3. G) Ribosomal operons (rrn, deemed essential) are not fragmented in P. distasonis, despite presence of ‘foreign DNA’ in vicinity. *, **, ***, for P<0.01, P<0.001, P<0.0001, respectively. Supplementary Table 4 shows rrn operons are not fragmented in other genera, i.e., Prevotella, Bacteroides, Alistipes, and Porphyromonas.

Figure 6.. Impact of ‘Foreign DNA’ insertions in Bacteroidota on metagenomics, antigenic operons and bacteria-bacteria inflammatory interactions in a model of gut microlesions.

A) Plot of DNA fragments that align between Parabacteroides CL11T00C22 and Bacteroides, Alistipes, Escherichia genomes (n=16) ordered by length of each aligning fragment. Notice genus-genus differences. B. thetaiotaomicron CL15T119C52 has unique pattern of abundant fragments (maximum fragment sizes and average % of genome shared, inset bar plots). B) BlastX (protein) and metagenomic (nucleotide) taxonomic analyses of 250bp-fragmented P. distasonis genome. Notice ‘extra species’ assigned by metagenomics (inflation), reflecting ‘foreign DNA’ insertions/exchange across Bacteroidota, and not real presence of species (inset bar plot). The n of ‘extra species’ varied with fragment length (Pearson corr. 0.84, P<0.05). The performance of BLAST and BLASTX depends on bacterial genome (Supplementary Figure 8). C) Metagenomic community simulation with Bacteroides, Parabacteroides, Alistipes, and Escherichia (1:1:1:1 genomes). Krona plot (relative abundances within hierarchies of metagenomic classifications (35)) illustrates E. coli sequences are poorly assigned to E. coli leading to relative ratio overestimation of Bacteroidota abundance (1:17:12:18; see Krona plots for individual genomes (‘Alone’) in Supplementary Figure 9). Bacteroides is commonly listed as ‘extra species’ (bar plot; complete list in Supplementary Figure 7) D) KEGG pathway and total gene counts in Enterobacteriaceae and Bacteroidota, highlighting the significant differences for Bacteroidota (details in Supplementary Table 7). E) Glycostaining of LPS extracted from E. coli and P. distasonis cultured in different media. F) Average pro-inflammatory cytokine secretion by bacteria in the stimulation, indicating that Bacteroidota release less pro-inflammatory cytokines compared to Enterobacteriaceae. G) Hypothetical model of gut microlesions with colonization of commensal/pathobionts modulating inflammation. Peritonitis model showed mice with Enterobacteriaceae had fatal peritonitis, but not if receiving Bacteroidota B. thetaiotaomicron, B. fragilis, or P. distasonis. *P<0.01; ****P<0.00001.