Two-week administration of engineered Escherichia coli establishes persistent resistance to diet-induced obesity even without antibiotic pre-treatment

Abstract

Adverse alterations in the composition of the gut microbiota have been implicated in the development of obesity and a variety of chronic diseases. Re-engineering the gut microbiota to produce beneficial metabolites is a potential strategy for treating these chronic diseases. N-acyl-phosphatidylethanolamines (NAPEs) are a family of bioactive lipids with known anti-obesity properties. Previous studies showed that administration of Escherichia coli Nissle 1917 (EcN) engineered with Arabidopsis thaliana NAPE synthase to produce NAPEs imparted resistance to obesity induced by a high-fat diet that persisted after ending their administration. In prior studies, mice were pre-treated with ampicillin prior to administering engineered EcN for 8 weeks in drinking water. If use of antibiotics and long-term administration are required for beneficial effects, implementation of this strategy in humans might be problematic. Studies were therefore undertaken to determine if less onerous protocols could still impart persistent resistance and sustained NAPE biosynthesis. Administration of engineered EcN for only 2 weeks without pre-treatment with antibiotics sufficed to establish persistent resistance. Sustained NAPE biosynthesis by EcN was required as antibiotic treatment after administration of the engineered EcN markedly attenuated its effects. Finally, heterologous expression of human phospholipase A/acyltransferase-2 (PLAAT2) in EcN provided similar resistance to obesity as heterologous expression of A. thaliana NAPE synthase, confirming that NAPEs are the bioactive mediator of this resistance.

Keywords: Antibiotics; Engineered bacteria; Gut microbiota; N-acyl-ethanolamides; N-acyl-phosphatidylethanolamines; N-acyltransferases.

Fig. 1

Biosynthetic and signaling pathways for N-acyl-ethanolamides (NAEs). Phosphatidylethanolamine (PE) N-acyltransferase enzymes transfer an acyl chain from a donor phospholipid to PE to form N-acyl-phosphatidylethanolamines (NAPE) which are then converted by NAPE hydrolyzing phospholipase D (NAPE-PLD) to NAEs, which in mammals act on receptors including PPARα, GPR119, GPR55, and TRPV1.

Fig. 2

Two-week treatment with pNAPE-EcN induces no effect in mice fed low fat diet, but induces persistent resistance to weight gain in mice fed a high fat diet, and pretreatment with antibiotics (Abx) does not enhance this effect. A. Gain in body weight from start of bacterial treatment for mice fed a low-fat chow diet. (Two-way ANOVA, p<0.0001 for time, p=0.8167 for treatment; B. Cumulative food intake for mice fed a chow diet. (Two-way ANOVA, p<0.0001 for time, p=0.0198 for treatment; Tukey’s multiple comparisons test ^p<0.05 Pretreatment Abx + pNAPE-EcN vs either Veh or pNAPE-EcN). C. Gain in body weight from start of bacterial treatment for mice fed a high fat diet. (Two-way ANOVA, p<0.0001 for time, p=0.0178 for treatment; Tukey’s multiple comparisons test *p<0.05 pNAPE-EcN vs Vehicle, #p<0.05 Abx+pNAPE-EcN vs Vehicle). D. Cumulative food intake for mice fed a high fat diet. (Two-way ANOVA, p<0.0001 for time, p=0.0307 for treatment; Tukey’s multiple comparisons test *p<0.05 pNAPE-EcN vs Vehicle, #p<0.05 Abx+pNAPE-EcN vs Vehicle). All values are shown as mean±SEM (n = 5 mice per group).

Fig. 3

Effect of antibiotic pretreatment and subsequent pNAPE-EcN treatment on fecal bacterial load and 16S microbiota composition of mice fed high fat diet A. Time course of fecal bacterial load. B. Heat map of 16S composition by phylum at experimental day 0, day 7 and day 16.

Fig. 4

Post-treatment administration of antibiotic cocktail eliminates pNAPE-EcN-induced reductions of weight gain, fat gain, and food intake in mice feeding on high fat diet. All values are mean±SEM (n = 6 mice per group). A. Effect of treatments on change in body weight from start of treatment (2way-ANOVA, treatment p < 0.0001, time p<0.0001, Tukey’s multiple comparison test ^# p<0.05 pNAPE-EcN+no Abx vs Vehicle or pEcN + Abx; *p <0.05 pNAPE-EcN+no Abx vs pEcN+Abx; ⁺p<0.05 pNAPE-EcN+Abx vs Vehicle or pEcN+Abx or pEcN+no Abx; ^ p<0.05 pNAPE-EcN+no Abx vs pNAPE-EcN+ Abx). B. Effect of treatments on change in body fat from start of treatment (2way-ANOVA, treatment p < 0.0001, time p<0.0001; Tukey’s multiple comparison test ^# p<0.05 pNAPE-EcN+no Abx vs Vehicle or pEcN+no Abx or pEcN + Abx; ^ p<0.05 pNAPE-EcN+no Abx vs pNAPE-EcN+ Abx). C. Effect of treatments on lean body mass (2way-ANOVA, p=0.9349 treatment, time p<0.0001). D. Effect of treatments on cumulative food intake from start of treatment (2way-ANOVA, treatment p<0.0001, time p <0.0001).

Fig. 5

Post-treatment administration of antibiotics (Abx) eliminates elevated NAPE levels. A. Fecal NAPEs on day 7 and day 21. B. Omental fat NAPE levels day 42. C. Plasma NAPE levels day 42. D. Plasma NAE levels day 42.

Fig. 6

Treatment with pPLAAT2-EcN is effective in reducing obesity in high-fat diet fed mice. All values are mean±SEM (n = 6 mice per group). A . Effect of treatments on gain in body weight from start of treatment (2way-ANOVA p = 0.0129 for treatment, p<0.0001 for time; Tukey’s multiple comparison test #p<0.05 for pPLAAT2-EcN vs Vehicle;*p<0.05 pPLAAT2-EcN vs EcN). B. Effect of treatments on gain of fat mass (2way-ANOVA, p < 0.0001 for treatment, p<0.0001 for time; Tukey’s multiple comparison test #p<0.05 for pPLAAT2-EcN vs Vehicle;*p<0.05 pPLAAT2-EcN vs EcN). C. Effect of treatments on lean body mass (2way-ANOVA, p=0.8205 for treatment, p<0.001 for time). D. Effect of treatments on cumulative food intake (2way-ANOVA, p=0.0001 for treatment, p <0.0001 for time; Tukey’s multiple-comparison #p<0.05 pPLAAT2-EcN vs Vehicle; *p<0.05 pPLAAT2-EcN vs EcN).)

Fig. 7

Treatment with pPLAAT2-EcN elevates fecal and tissue NAPE levels at 4 weeks post-treatment. A. Total NAPE levels in feces collected on day 41 from start of treatment. (1way ANOVA p<0.0001; Tukey’s multiple comparison test *p<0.05 pPLAAT2-EcN vs Vehicle or EcN). B. Total NAPE levels in omental fat pads (1way ANOVA p<0.0001; Tukey’s multiple comparison test *p<0.05 pPLAAT2-EcN vs Vehicle or EcN). C. Total NAPE levels in plasma (1way ANOVA p=0.0005; Tukey’s multiple comparison test *p<0.05 pPLAAT2-EcN vs Vehicle or EcN). D. Total NAE levels in plasma (1way ANOVA p<0.0336; Tukey’s multiple comparison test *p<0.05 pPLAAT2-EcN vs Vehicle). All values are mean±SEM, n=6.