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. 2020 Feb 18;30(7):2297-2305.e5.
doi: 10.1016/j.celrep.2020.01.078.

"V体育安卓版" Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli

Michael J Ormsby  1 , Síle A Johnson  1 , Nuria Carpena  1 , Lynsey M Meikle  1 , Robert J Goldstone  2 , Anne McIntosh  1 , Hannah M Wessel  1 , Heather E Hulme  1 , Ceilidh C McConnachie  1 , James P R Connolly  3 , Andrew J Roe  1 , Conor Hasson  1 , Joseph Boyd  1 , Eamonn Fitzgerald  1 , Konstantinos Gerasimidis  4 , Douglas Morrison  5 , Georgina L Hold  6 , Richard Hansen  7 , Daniel Walker  1 , David G E Smith  2 , Daniel M Wall  8
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"VSports最新版本" Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli

"V体育官网入口" Michael J Ormsby et al. Cell Rep. .

Abstract

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients VSports手机版. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence. .

Keywords: Crohn's disease; adherent-invasive E. coli; propionic acid; short chain fatty acid V体育安卓版. .

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Conflict of interest statement

Declaration of Interests R. H VSports最新版本. has received conference travel expenses and consultancy fees from Nutricia-Danone and 4D Pharma. K. G. received research grants and speakers fees from Nestle Health Sciences, Nutricia-Danone, and Mylan.

Figures

None
Graphical abstract
Figure 1
Figure 1
Supplementation of the Murine Intestine with PA Raises the PA Concentration, and Infection of This Model with AIEC Increases the Virulent Phenotype of the Bacterium (A) The ratio of acetate:propionate:butyrate in murine samples was calculated from the isolated caecal contents of an uninfected control mouse that was mock infected with PBS in place of bacteria, 3 days post-treatment with exogenous PA added to the drinking water. This was compared to published human SCFA ratios (Cummings et al., 1987). (B and C) AIEC type strain LF82 modified to contain the luciferase and erythromycin cassette (LF82lux) was recovered from mice that had been given water (−PA) or water supplemented with 20 mM propionic acid (+PA). Subsequently, adhesion to Caco-2 intestinal epithelial cells (B) and ability to form biofilms in RPMI with 3% fetal calf serum (FCS) (C) were assessed. In vitro/vivo refers to where the strains were generated. Results displayed are the average of at least three independent biological replicates ± SD. Samples were analyzed using an un-paired non-parametric t test (A) or one-way ANOVA with Holm-Sidak’s multiple comparisons post-test (B and C; ∗p < 0.05; ∗∗p < 0.01).
Figure 2
Figure 2
LF82 Utilizes PA as a Sole Carbon Source for Growth, and Exposure to PA Increases Virulence (A) LF82 and commensal E. coli strain F-18 were grown in minimal media supplemented with 20 mM PA over a series of five successive re-cultures. Growth rate of the PA-exposed strains, termed LF82-PA and F-18-PA, were subsequently compared to WT controls. (B) The growth rates of LF82, F-18, LF82-PA, and F-18-PA were unchanged in rich LB broth. (C and D) The ability of LF82 and LF82-PA strains to adhere to (C) and invade (D) Caco-2 intestinal epithelial cells was determined. Biofilm formation over 7 days of anaerobic growth was assessed in the presence of 20 mM PA (D). (E) The ability of LF82 and LF82-PA to tolerate acidic pH (pH 3) over time was determined by colony counts (E). Results displayed are the average of at least three independent biological replicates ± SD. Samples were analyzed using an unpaired t test where ∗p < 0.05 and ∗∗p < 0.01 (C–F).
Figure 3
Figure 3
The Enhanced Fitness and Virulence of the AIEC-PA Phenotype Can Be Reversed AIEC type strain, LF82, and clinical isolates B94, B115, B122, and B125 were repeatedly cultured in minimal media with 20 mM PA as the sole carbon source. These isolates were then continuously re-cultured (5 passages) in LB, generating a reverted strain (termed Isolate-PA-LB). Adhesion (A) and invasion (B) to Caco-2 intestinal epithelial cells were examined. Data displayed are of two independent experimental replicates, with each experiment including three independent biological replicates. Adherence and invasion data are expressed as mean ± SD; data were analyzed using a one-way ANOVA with Tukey post-test correction (∗p < 0.05; ∗∗p < 0.01).
Figure 4
Figure 4
Global Transcriptional Shifts of LF82-PA (A) Volcano plot illustration of gene expression between LF82 and LF82-PA, as determined by RNA-seq. Significance (Log10 p value) and fold change cutoffs (log2) are indicated by the dashed and solid lines, respectively. (B) Significantly differentially expressed genes (DEGs) are numbered and highlighted in green (upregulated in LF82-PA) and red (downregulated). (C) Genes corresponding to ribosomal RNA coding regions were not labeled for clarity. Bar chart highlighting the fold changes in expression of the identified DEGs. Each gene is numbered and corresponds to the volcano plot. DEGs are grouped into functional categories.
Figure 5
Figure 5
Pre-exposure of AIEC to PA Combined with Exogenous PA Supplementation Promote Colonization and Long-Term Persistence Drinking water was supplemented where indicated with 20mM PA and provided to male C57BL/6 mice for 3 days prior to infection. Persistence of LF82 (A and B) and LF82-PA (C and D) was determined in the ileum (A and C) and colon (B and D) 21 days post-infection by colony counts. Data are expressed as CFU/gram of tissue ± SD and were analyzed using an unpaired t test (∗p < 0.05).
Figure 6
Figure 6
Competitive Index of LF82 from PA-Fed Mice versus LF82 from Water-Fed Mice For the primary infection (1°), one group of mice (n = 4) were fed 20 mM PA for 3 days prior to challenge. A second group of mice were given only sterile water. A subset of each of these mice were then infected with either LF82 or LF82 carrying a luciferase and erythromycin cassette (LF82lux), and the infection was allowed to proceed for 7 days. This resulted in the recovery of strains labeled LF82-PA1°, LF82-W1°, LF82lux-PA1°, and LF82lux-W1° from ileal and colonic homogenates. Bacteria were isolated using LB supplemented with either ampicillin or erythromycin. Subsequently, equal quantities of LF82-PA1° and LF82lux-W1° were mixed, as were LF82lux-PA1° and LF82-W1°, giving final concentrations of 1 × 109 CFU mL−1 (0.5 × 109 CFU mL−1 of each strain). The ratios of LF82-PA1°:LF82lux-W1° and LF82lux-PA1°:LF82-W1° were determined by plating on LB plates supplemented with ampicillin or erythromycin. Mice were again given either 20 mM PA or sterile drinking water for 3 days prior to challenge, before infection with either LF82-PA1°:LF82lux-W1° or LF82lux-PA1°:LF82-W1°. Seven days post-infection, bacteria were recovered by plating ileal and colonic homogenates on either ampicillin or erythromycin. Competitive indices (CI) were determined by normalization to the initial inoculum ratios. Black circles represent LF82-PA1°, whereas white circles represent LF82lux-PA1°. The solid black line at CI = 1 represents LF82-W1° and LF82lux-W1°. Statistical analyses for each dataset were conducted using the individual colony counts. A two-tailed Wilcoxon rank-sum test was conducted (∗p < 0.05 was deemed significant).
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