Bile acid fitness determinants of a Bacteroides fragilis isolate from a human pouchitis patient

Abstract

The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.

Keywords: Bacteroides fragilis; Tn-seq; bile; cell envelope; deoxycholate; pouchitis; stress response.

Fig 1

B. fragilis P207 growth in the presence of increasing concentrations of deoxycholate (DC), bile salt mixture (BSM), or bile extract from porcine (BEP). Data reflect the terminal density (optical density at 600 nm of 3-mL cultures after 24-hour growth for DC and BSM or 48 hours for BEP; mean ± SD of eight independent trials, each normalized to an untreated control. Representative growth curves for each condition are presented in Fig. S1. Blue arrow highlights the concentration of DC and BSM used for subsequent experiments. Black arrows highlight the concentrations of BEP used for subsequent experiments.

Fig 2

Treatment of B. fragilis P207 with a sub-lethal concentration of deoxycholate (DC) induces large-scale activation and repression of transcription. Volcano plots of differentially transcribed genes at (A) 6 minutes and (B) 20 minutes after DC exposure. Lines indicate cutoff criteria {FDR P < 10⁻¹⁰ and absolute log₂[fold change (FC)] >1.5, where FC reflects the ratio of counts per million [CPM] after DC exposure/CPM before DC exposure}. The number of up- or down-regulated transcripts (blue points) at each time point is indicated at the bottom of the graph (blue text). Genes that do not meet the cutoff criteria are in black. Functional categories of genes, highlighted with special symbols, include (i) translation processes (GO terms: 0003735, 0006414, 0006400, 0043022, and 0000049; orange diamonds); (ii) transmembrane efflux processes (operon PTOS_003611–3614, red stars); (iii) unfolded protein stress (GO term: 0051082, pink triangles); (iv) redox stress (catalase, msrB, and dps; purple hexagons); (v) tRNA (related to translation processes, yellow inverted triangles). A subset of transcriptional changes was confirmed with reverse transcription quantitative PCR (Fig. S2). (C) Transcript abundance 6 minutes (orange circles) and 20 minutes (blue circles) after 0.01% (wt/vol) DC exposure is highly correlated. The mean CPM at each time point for each of the 1,071 genes that are significantly up- and down-regulated by 20 minutes after DC treatment are plotted, ranked by CPM in the untreated condition (black circles).

Fig 3

Genes that contribute to B. fragilis fitness in the presence of purified bile acids and crude bile have diverse metabolic, stress response, and cell envelope functions. Heat map of fitness scores for the 122 genes that are significant determinants of B. fragilis fitness in at least one treatment condition (see Materials and Methods for significance thresholds); treatment conditions are arranged in columns and genes are arranged in rows. Composite fitness scores for mutants grown in plain BHIS medium without bile are labeled (plain). Mutant strain growth was measured in BHIS containing 0.01% (wt/vol) bile salt mixture (BSM), 0.01% (wt/vol) deoxycholate (DC), and 0.04%, 0.08%, and 0.16% bile extracts from porcine (BEPs). Gene-level fitness scores were hierarchically clustered; gene arrangement was manually adjusted to group genes presumed to be in operons. White blocks indicate genes with insufficient barcode counts in the reference condition of a particular experiment to calculate a fitness score. Colored gene names highlight select functional categories discussed in the text: red, efflux systems; light green, V-ATPase operon; dark green, cardiolipin synthase genes; light blue, lipid metabolism; mustard, central carbon metabolism; orange, bat aerotolerance operon; fuchsia, plasminogen-binding protein; and purple, gmd protein glycosylase.

Fig 4

Genes that are transcriptionally regulated by DC treatment are weakly correlated with genes that determine fitness in a medium containing DC. Log₂(fold change) in transcript levels before and after 20 minutes of exposure to 0.01% DC (x-axis) versus gene-level fitness scores after cultivation in 0.01% DC (y-axis) for the 3,270 genes with fitness scores. Each point is a gene for (A) all genes with fitness scores, (B) transcriptionally regulated genes with fitness data, (C) genes that pass fitness criteria, or (D) transcriptionally regulated genes that also pass fitness criteria. The 25 genes in panel D are listed in Fig. S5. Dotted lines indicate cutoff thresholds on each axis. Numbers on the graph indicate the number of genes in each region of the graph that meets the selection criteria.

Fig 5

Fitness scores highlight metabolic branch points in central carbon and lipid metabolism. (A) Schematic of glucose metabolism pathways highlighting the branch point between glycolysis and the pentose phosphate pathway. (B) Schematic of lipid metabolism highlighting the fate of long-chain fatty acids (LCFAs). Blue and red arrows and enzyme circles highlight processes that promote or inhibit fitness in the presence of DC, respectively. Pathway names are bold; key enzymes are named in circles; metabolites are in plain text. (C) Heat map of expression values and fitness scores for the genes highlighted in panels A and B. Transcript abundance is presented as reads per kilobase per million reads (RPKM), and regulation is indicated by the log₂(fold change) compared to untreated cells (0 minute). Color scales representing absolute expression, fold change in expression, and mutant fitness scores are presented below each type of data.

Fig 6

B. fragilis (Bf) encodes two rotary ATP synthase/ATPase systems; the V-type system bears conserved residues for sodium ion translocation and is a critical fitness factor in deoxycholate. (A) Expression level and fitness scores for genes in the V-type and F-type ATP synthase/ATPase operons, presented as in Fig. 5. White blocks indicate genes with insufficient barcode counts in the reference condition to calculate a fitness score. (B) Operon structure of F-type and V-type ATPase systems. PTOS gene locus numbers are below gene outlines; annotated sub-unit names are in white. Genes are colored as follows: blue, cytoplasmic ATP synthesis/hydrolysis sub-units; red, transmembrane, ion-translocating, rotary sub-units; orange, transmembrane stator sub-units; green, stalk sub-units connecting the enzymatic complex to the membrane complex; and gray, unknown function. The V-type system has fewer connecting accessory sub-units than the F-type system or other characterized bacterial V-type systems (66). (C) Protein sequence alignment of the K sub-units of Bf and the sodium ion-translocating Enterococcus hirae (Eh) V-ATPases. Identical residues are in bold. Residues experimentally determined to coordinate sodium ions in E. hirae (65) are highlighted in blue with asterisk.