Escherichia coli Affects Expression of Circadian Clock Genes in Human Hepatoma Cells

Abstract

Recent research has indicated that dysbiosis of the gut microbiota can lead to an altered circadian clock of the mammalian host. Herein we developed an original system that allows real-time circadian studies of human HepG2 hepatoma cells co-cultured with bacteria. The HepG2 cells with stably integrated firefly luciferase reporter under the control of PERIOD2 promoter were co-cultured with E. coli strains isolated from human fecal samples from healthy individuals. The two E. coli strains differ in the phylogenetic group and the number of ExPEC virulence-associated genes: BJ17 has only two, and BJ23 has 15 of 23 tested. In the first 24 h, the E. coli BJ17 affected the HepG2 circadian clock more than BJ23. Cosinor analysis shows a statistically significant change in the amplitude of PER1 and 2 and the phase advance of PER3. A high percentage of necrotic and apoptotic cells occurred at 72 h, while a correlation between the number of ExPEC genes and the influence on the HepG2 core clock gene expression was observed. Our study reveals that the E. coli genetic background is important for the effect on the mammalian circadian clock genes, indicating possible future use of probiotic E. coli strains to influence the host circadian clock.

Keywords: Escherichia coli; HepG2; circadian clock; circadian rhythm; intestinal microbiota.

Figure 1

HepG2 PER2-dluc cell line activity measured with Lumicycle. Co-cultures with bacterial strain BJ17 (white dots) or BJ23 (grey dots) and control HepG2 PER2-dluc cells without bacteria (black dots). Data are represented as the mean of two separate experiments.

Figure 2

Comparison of circadian behavior of core clock gene expression between the co-cultures and control. The figures represent the relative RNA expression of the core clock genes as a function of time (2–24 h) in HepG2 cells (K, black dots and lines, respectively) and in HepG2 cell co-cultures with E. coli strains BJ17 and BJ23 (red dots and lines, respectively). The experimental results, obtained in two separate experiments, are represented with dots, and the fitted cosinor curves with solid lines. The raw data used in the fitting process are available in Tables S2–S22. The quantitative results obtained with the cosinor analysis are available in Tables S1 and S23.

Figure 3

Relative expression of the core circadian genes as a function of time (24–72 h). BJ17 (white squares), BJ23 (grey triangles): co-cultures of BJ strain with the HepG2 cells. Control (black circles): HepG2 cells without bacteria. Statistically significant results were presented; not significant (ns) for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001 and **** for p ≤ 0.0001. Data are represented as mean ± SD of two separate experiments.

Figure 4

Results of apoptosis/necrosis test. Percentages of live cells (light grey), late apoptotic cells (dark grey), early apoptotic (white) and necrotic (black) cells are represented as mean ± SD of two separate experiments.