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

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.


Introduction
Gut microbiota has been extensively studied in recent years, and multiple features associated with human health have been discovered. It is now clear that the bacteria in our intestine impact host metabolism [1,2], prevent colonization by pathogens [3], affect the immune system [4,5] and through all these actions help to maintain the homeostasis of the host. An "imbalance" of the microbiota, the so-called "dysbiosis", has been associated with different diseases, ranging from bowel diseases to psychiatric disorders [6]. Moreover, the gut microbiota interferes with the circadian clock of the host [1,2,[7][8][9][10]. While it became accepted that microbiota can modulate the host's core clockwork machinery, we know very little about the influence of individual bacterial strains.
The circadian system consists of the central circadian clock (=central pacemaker) located in the hypothalamus, more precisely in the suprachiasmatic nucleus (SCN), and peripheral clocks in almost every mammal cell and tissue. There are many cues, such as light, food intake, metabolites and sleep cycle that orchestrate the circadian system. On the molecular level, circadian rhythms are maintained by two interlocking transcriptiontranslation feedback loops. The core loop consists of transcriptional activators circadian associated genes and another with only a few such genes, in light of their potential to interfere with the mammalian hepatic circadian clock.

Experimental Model and Subject Details
Preparation of Human Hepatoma cells with stably integrated PER2-dluc. HepG2 cells were obtained from ATCC (HB-065) and kept stored in liquid nitrogen until used. The cells were cultured in a monolayer in Dulbecco's Modification of Eagle's Medium (DMEM, Sigma Aldrich, Taufkirchen, Germany, SI-D6429) supplemented with a 10% fetal bovine serum (FBS, Sigma Aldrich) and 1% penicillin/streptomycin (PS, Sigma Aldrich) antibiotic mix at 37 • C in an atmosphere of 5% CO 2 . When the cells reached 70-80% confluency, they were trypsinized, resuspended in fresh media and used in further experiments.
To enable real-time monitoring of circadian cell behavior with the LumiCycle apparatus 32 (Actimetrics, Wilmette, IL, USA), a new stable cell line transfected with PER2-dluc plasmid was prepared. PolyJet™ In Vitro DNA Transfection Reagent (SignaGen, Frederick, MD, USA) and the PolyJet TM advanced protocol for transfecting mammalian cells was used for stable introduction of PERIOD2 promoter controlling the Luciferase reporter (PER2-Luc) into the genome of HepG2 cells. The protocols were performed according to the manufacturer's instructions. Further, the selection of antibiotic-resistant clones and single cell cloning was performed in 96 well plates. Clones with the best circadian parameter characteristics as determined on LumiCycle were chosen for further analysis.

Bacterial Growth Curves
For the determination of bacterial growth curves, bacterial strains were grown in liquid LB media overnight at 37 • C with aeration (5% CO 2 ). The next day, overnight cultures were diluted 1:100 in fresh, pre-warmed LB media in a conical flask and grown for 7 h. Each hour, samples were taken for measurement of optical density at 600 nm (OD600) and for determination of colony-forming units per milliliter (CFU/mL) on LB plates. Before transfer onto the LB plate, samples were 10-fold serial diluted (Table S24) in PBS. Data were obtained from two independent experiments performed in duplicate.
Moreover, the data obtained in these growth curve experiments were used in linear regression analysis ( Figure S1B) to estimate the relationship between OD600 and CFU/mL in order to be able to calculate the needed amount of bacteria for multiplicity of infection (MOI) 0.005:1 (bacteria:HepG2 cell) in co-culture experiments with HepG2 cell line.

Stable Co-Culture Preparation
Bacterial strains were transferred from LB plates stored at 4 • C to LB media and grown overnight at 37 • C and 5% CO 2 . The next day, bacterial cultures were diluted 1:100 in fresh, pre-warmed LB media and incubated for 2.5-3 h to reach the early exponential phase. Based on the OD600 measurements, the needed amount of bacteria for MOI 0.005 was resuspended in recording media (200 µM L-glutamine (Gibco, Waltham, MA, USA), 100 µM Na-pyruvate (Gibco, Waltham, MA, USA), 10% FBS, 4 % d-luciferin (Sigma Aldrich, Taufkirchen, Germany) and DMEM: high glucose, no glutamine, no phenol red) and transferred onto synchronized HepG2 PER2-dluc cells cultured on 34 mm dishes. Dishes were sealed with parafilm and placed in LumiCycle apparatus for 24 h (37 • C, 5% CO 2 ). Each experiment was performed in duplicate on two 35 mm Petri dishes.

72 h Sampling
Bacterial cultures used to infect the HepG2 cells were prepared as described in Stable co-culture preparation, with the exception that they were finally resuspended in DMEM supplemented with 10% FBS. The bacterial suspension was then transferred on synchronized HepG2 cells cultured in 24-well plates. Co-culture was incubated for 4 h. Then, bacteria were discarded and cells were washed 3 times with PBS before DMEM supplemented with a 10% FBS and 1% penicillin/streptomycin antibiotic mix was applied. Timepoint 0 was determined as the moment when bacteria were discarded. Sampling was performed every 2 h for 24 h, with additional time points at 48 h and 72 h. Samples, obtained in two independent experiments-two separated wells on the same plate and performed in triplicate-were stored at −80 • C until the isolation of total RNA.

RNA Isolation and RT-PCR Analysis
RNA from HepG2 cells was extracted by Trizol reagent (Sigma Aldrich) following manufacturer's instructions. Quality and quantity of RNA were determined using Nan-oDrop™ (ThermoFisher Scientific, Waltham, MA, USA), diluted accordingly and after DNA removal with DNase I Recombinant Kit (Roche, Basel, Switzerland) transcribed to cDNA with Transcriptor Universal cDNA Master (Roche) according to the manufacturer's protocols. PCR analysis was performed on Lightcycler 480 using Sybr Green Master Mix (Roche, Basel, Switzerland) kit according to manufacturer's protocol. Primers used are listed in Table S25. Target gene expression was normalized on reference genes Rplp0 and ActB, appraising also amplification efficiency of a qPCR.

Apoptosis-Necrosis Assay
For the apoptosis-necrosis assay, FITC Annexin V Apoptosis Detection Kit II (cat. num. 556570, BD Pharmigen) was used. Time points 0, 24, 48 and 72 h were chosen for this assay. Non-treated cells were used as negative control. After incubation (0, 24, 48 or 72 h), cells were washed with PBS and harvested by trypsinization (Trypsin (Sigma Aldrich), 1 min. incubation), centrifuged at 500× g for 5 min, and washed with 1% BSA (Sigma Aldrich) in PBS. Then, a sample for flow cytometry analysis was prepared according to manufacturer protocol. Each sample was filtered through polystyrene tube with strained cap and analyzed by the machine BD FACSCanto II System (BD Biosciences, Franklin Lakes, NJ, USA) with the BD FACSDiva™ Software (BD Biosciences, Franklin Lakes, NJ, USA). The assay was performed in duplicates.

Quantification and Statistical Analysis
Microsoft Excel was used for calculation of the mean ±SD and to perform the linear regression analysis. For comparison analysis of circadian genes expression at time points Rhythmicity analysis of observed circadian genes was performed with a singlecomponent cosinor model [39], for which a 24 h period was presumed. This model can be described as where X 1 = sin T 24 ·2π and X 2 = cos T 24 ·2π , and where T presents the vector of time points in which measurements were obtained. We fitted the measured data to this model using linear regression. The obtained models were used to assess the circadian parameters of the data and their significance. We evaluated the amplitudes and acrophases using Equations (2) and (3), respectively.
We evaluated the significance of rhythmicity of fitted data with zero-amplitude test using the F-statistic as described in Bingham [40].
An extended single-component cosinor model can be used to assess the differential rhythmicity among pairs of different circadian experiments: where parameter g equals 0 if the data belong to the first experiment, and 1 if the data belong to the second experiment, respectively. We applied this model to different pairs of experiments, namely control (K) vs. perturbation (BJ17 or BJ23), to assess the difference of circadian parameters between the experiments (amplitude change and phase shift) as well as their significance [40]. Rhythmicity and differential rhythmicity analyses were implemented in Python 3 using a recently developed CosinorPy package [41]. The reported significance values are adjusted for multiple testing using the Benjamini and Hochberg procedure.

Assessing Growth Curves of E. coli Strains BJ17 and BJ23
The characteristics of both E. coli bacterial strains are described in the Microbial strains section. Growth characteristics of BJ17 strain (low number of ExPEC virulence-associated genes) and BJ23 strain (high number of ExPEC virulence-associated genes) were evaluated during a 7 h incubation as described in the Bacterial Growth Curves section. The results ( Figure S1A) show similar growth curves for both bacterial strains, as determined by measurement of optical density at 600 nm (OD600) and determination of colony-forming units per milliliter (CFU/mL) on LB plates. The bacterial growth data obtained in the exponential phase (2-5 h) were analyzed with linear regression ( Figure S1B). The obtained ratio OD600 to CFU/mL was used in further experiments to determine the needed amount of bacteria for multiplicity of infection (MOI) 0.005 in co-culture.

Co-Culture of HepG2 Cells with Bacterial Strains Is Stable for 4 h
HepG2 cells with stably integrated firefly luciferase reporter under control of the PERIOD2 promoter (HepG2 PER2-dluc) were inoculated with either BJ17 or BJ23 E. coli Microorganisms 2021, 9, 869 6 of 14 strain, and their co-culture was followed for 25 h in real time with the Lumicycle apparatus. As seen in Figure 1, after 4 h, the signal of the measured luminescence in co-cultures decreased, while the luminescence signal of the control, HepG2 PER2-dluc cells without bacteria, was still increasing. From this, we concluded that both co-cultures are stable for 4 h, and therefore, in all following experiments, the incubation of the co-cultures was always 4 h.

Co-Culture of HepG2 Cells with Bacterial Strains is Stable for 4 h
HepG2 cells with stably integrated firefly luciferase reporter under control of the PE-RIOD2 promoter (HepG2 PER2-dluc) were inoculated with either BJ17 or BJ23 E. coli strain, and their co-culture was followed for 25 h in real time with the Lumicycle apparatus. As seen in Figure 1, after 4 h, the signal of the measured luminescence in co-cultures decreased, while the luminescence signal of the control, HepG2 PER2-dluc cells without bacteria, was still increasing. From this, we concluded that both co-cultures are stable for 4 h, and therefore, in all following experiments, the incubation of the co-cultures was always 4 h.

The 4 h Incubation of HepG2 Cells with E. coli affects Expression of HepG2 Circadian Genes
After the 4 h incubation of HepG2 cells with bacterial strains, the bacteria were removed, and the HepG2 cells were prepared for the 72 h sampling assay in order to reveal the influence of applied E. coli on the hepatic HepG2 circadian clock. HepG2 cells were sampled every 2 h during the first 24 h and then two more samples were collected, one at 48 h and the other at 72 h. The central genes of the circadian clock, transcriptional activators CLOCK and BMAL1, and repressors PER1, PER2, PER3, CRY1 and CRY2 were studied and statistical analyses were performed. The first 24 h were analyzed with the cosinor test. The results in Figure 2 show the core clock gene expression in HepG2 cells grown in the absence (control-K) or presence of E. coli strains BJ17 and BJ23. In control HepG2 cells, the statistical tests (Table S1) confirmed the circadian expression of the genes encoding circadian repressors PER1,2,3 and CRY1,2, while we failed to detect the circadian wave for the activator proteins CLOCK and BMAL1. The addition of bacteria retained the rhythmic expression of HepG2 core clock genes except for CRY1 and enhanced the circadian behavior of CLOCK and BMAL1. Taken together, the measured core clock genes, with the exception of CRY1, were expressed rhythmically also after being exposed to E. coli strains.

The 4 h Incubation of HepG2 Cells with E. coli Affects Expression of HepG2 Circadian Genes
After the 4 h incubation of HepG2 cells with bacterial strains, the bacteria were removed, and the HepG2 cells were prepared for the 72 h sampling assay in order to reveal the influence of applied E. coli on the hepatic HepG2 circadian clock. HepG2 cells were sampled every 2 h during the first 24 h and then two more samples were collected, one at 48 h and the other at 72 h. The central genes of the circadian clock, transcriptional activators CLOCK and BMAL1, and repressors PER1, PER2, PER3, CRY1 and CRY2 were studied and statistical analyses were performed. The first 24 h were analyzed with the cosinor test. The results in Figure 2 show the core clock gene expression in HepG2 cells grown in the absence (control-K) or presence of E. coli strains BJ17 and BJ23. In control HepG2 cells, the statistical tests (Table S1) confirmed the circadian expression of the genes encoding circadian repressors PER1,2,3 and CRY1,2, while we failed to detect the circadian wave for the activator proteins CLOCK and BMAL1. The addition of bacteria retained the rhythmic expression of HepG2 core clock genes except for CRY1 and enhanced the circadian behavior of CLOCK and BMAL1. Taken together, the measured core clock genes, with the exception of CRY1, were expressed rhythmically also after being exposed to E. coli strains.
An important aspect in evaluating whether bacteria affected the inner clock of mammalian cells is the estimation of circadian parameters (Table S23). A statistically significant change of the amplitude, compared to controls, was observed for HepG2 genes PER1 (decrease) and PER2 (increase) grown with E. coli BJ17. The change in the phase of gene expression (phase shift) was observed only for PER3, again grown with E. coli BJ17. The rhythmic expression of PER3 was advanced by 4.23 h, meaning that the expression of this circadian repressor was phase-advanced compared to other core clock genes.
To evaluate whether the 4 h co-culture of the E. coli strains affects the long-term expression of the core clock genes, we monitored their expression at 24 h, 48 h and 72 h (Figure 3). It is evident that both E. coli strains diminish the expression of the core clock genes after 72 h. The relation between the number of ExPEC virulence-associated genes and the influence on the circadian clock of HepG2 cells is obvious. The cells co-cultured with E. coli BJ23 with more virulence factors in most cases show a lower expression of core clock genes compared to cells co-cultured with E. coli BJ17. Exceptions are the transcriptional activator BMAL1 and the repressor PER3, where there is no statistical difference between the bacterial strains.

The 4 h Incubation of HepG2 Cells with the Bacterial Strain with More Virulence-Associated Genes Did Affect the HepG2 Cells Viability
The viability of HepG2 cells was determined by the apoptosis/necrosis assay and flow cytometry. After the 4 h incubation of HepG2 cells with bacterial strains, the bacteria were removed and the apoptosis/necrosis assay on HepG2 cells was performed at 0 h (immediately after removal of the bacteria), 24 h, 48 h and 72 h after bacterial removal. The results (Figure 4) showed that more HepG2 cells were apoptotic and necrotic in co-culture with E. coli strain BJ23 possessing more ExPEC virulence-associated genes. Importantly, the viability of HepG2 cells in the co-culture with BJ17 strain was similar to controls, where HepG2 cells were grown without bacteria (Figure 4). Figure 4 shows also that the largest percentage of apoptotic and necrotic cells in both co-cultures was at time point 0 h.

The 4 h Incubation of HepG2 Cells with the Bacterial Strain with More Virulence-Associated Genes Did Affect the HepG2 Cells Viability
The viability of HepG2 cells was determined by the apoptosis/necrosis assay and flow cytometry. After the 4 h incubation of HepG2 cells with bacterial strains, the bacteria were removed and the apoptosis/necrosis assay on HepG2 cells was performed at 0 h (immediately after removal of the bacteria), 24 h, 48 h and 72 h after bacterial removal. The results (Figure 4) showed that more HepG2 cells were apoptotic and necrotic in co-culture with E. coli strain BJ23 possessing more ExPEC virulence-associated genes. Importantly, the viability of HepG2 cells in the co-culture with BJ17 strain was similar to controls, where HepG2 cells were grown without bacteria (Figure 4). Figure 4 shows also that the largest percentage of apoptotic and necrotic cells in both co-cultures was at time point 0 h.

Discussion
The circadian clock was most recently recognized as the essential molecular link between the physiology of the mammalian host and microorganisms. There is more evidence that the host mammalian circadian clock shapes the microbiome [42]. However, data about bidirectional communication between microorganisms and their hosts is increasing [5]. We are only in the early phases of understanding how the microbiota, and its metabolic outputs affect the rhythmic processes, such as immunity [42]. Microbiota can

Discussion
The circadian clock was most recently recognized as the essential molecular link between the physiology of the mammalian host and microorganisms. There is more evidence that the host mammalian circadian clock shapes the microbiome [42]. However, data about bidirectional communication between microorganisms and their hosts is increasing [5]. We are only in the early phases of understanding how the microbiota, and its metabolic outputs affect the rhythmic processes, such as immunity [42]. Microbiota can influence the host's circadian gene expression in peripheral tissues by the produced metabolites, such as short-chain fatty acids and unconjugated bile acids [43]. As most results regarding the host-microbiome circadian interactions originate from mouse models, it is also essential to obtain verification results in human-derived cells and tissues.
Herein we developed an original system that allows real-time circadian studies of human hepatoma cells co-cultured with bacteria. Hepatocytes are key cell types for innate immunity [35] and therefore they represent an interesting model to study bacteria-human host interaction. Cell line HepG2 has been used in previous studies to demonstrate the preventive effect of drug candidates on circadian rhythm disorders [30,31]. The HepG2 cells with stably integrated firefly luciferase reported under the PERIOD2 promoter were co-cultured with two E. coli strains isolated from human fecal samples obtained from healthy individuals. These bacterial strains differ in the phylogenetic group and number of present ExPEC virulence-associated genes. The BJ17 strain can be designated as a commensal E. coli strain as it belongs to the A phylogenetic group, which is associated with commensal strains [15] and has only two of the tested ExPEC virulence-associated genes, both belonging to the iron uptake systems (fyuA of the yersiniabactin and iroN of the salmochelin iron uptake system). The BJ23 strain can be designated as a potential ExPEC strain, as it belongs to the phylogenetic group B2, which is associated with ExPEC strains [15] and possesses 15 out of 23 tested ExPEC virulence-associated genes, including the ibeA encoding the invasin IbeA and clbAQ associated with the genotoxin colibactin. Based on the results obtained from the Lumicycle experiment (Figure 1), it can be concluded that HepG2 co-cultures with both E. coli strains (BJ17-HepG2 PER2-dluc and BJ23-HepG2 PER2-dluc) are stable for 4 h.
We were interested to examine whether the HepG2 core circadian clock was influenced by the bacteria in line with their number of ExPEC virulence-associated genes. We tested the hypothesis of whether bacteria with more ExPEC virulence-associated genes have a stronger effect on the host's circadian clock. A simple statistical analysis of the core clock gene expression at 24 h, 48 h and 72 h showed that, only after 72 h, the relation between the number of ExPEC virulence-associated genes and the influence on the core circadian clock gene expression of HepG2 cells can be confirmed (Figure 3). In the first 24 h after co-culture of HepG2 cells with E. coli, the E. coli BJ17 seems to affect the circadian clock more than E. coli BJ23 (Figure 2). The cosinor analysis with adjusted p-value shows a statistically significant change in the amplitude of PER1 and PER2, and the phase advance of PER3, all in the case when HepG2 cells were co-cultured with the less virulent E. coli BJ17. In the literature, we can see that pathogen-associated molecular patterns caused several modulations of the clock gene expression. In particular, researchers showed that the synthetic triacylated lipopeptide Pam3CSK4, and the ODN 1826, a DNA rich with CpG motifs as it is common for bacterial DNA, caused increased mRNA levels of PER2 [10]. Likewise, we could observe an increase in the PER2 amplitude of HepG2 in contact with both strains; in the case of BJ17, the increase was found to also be statistically significant. Literature reports that certain bacteria can also affect Bmal1 expression: Helicobacter pylori activates transcription of LIN28A in gastric tissue, which binds to the BMAL1 promotor. In this way, H. pylori infection upregulates the transcription of BMAL1 and dysregulates the molecular clock in gastric tissues. Enhanced levels of BMAL1 stimulate the expression of pro-inflammatory cytokine TNF-α and promote inflammation [44]. On contrary, BMAL1 was downregulated in hepatocytes at Staphylococcus oralis infection, and it affected also coagulation processes in the liver [32]. In our case hepatocytes co-cultured with E. coli enhanced amplitude of expression of BMAL1 compared to control, although not significantly. Thus, it would be tempting to speculate that bacterial infection affects the expression of circadian genes, but reaction depends on host tissue and the properties of bacteria, but, so far, this hypothesis has not been tested.
In addition, LPS, an endotoxin commonly found in the outer membrane of Gramnegative bacteria, has been shown to modify circadian clock genes. LPS changed the expression of core circadian genes and linked immune pathways. LPS injection suppressed expression of circadian genes: Per1, Per2, Dbp, PPARα, and rFKBP51 mRNA in rats liver [45]; Per2 in mouse ovary [46], in macrophages [47] and synovial cells [48]; Per1 and Per2 in hearth and liver [49]. Based on these findings, Per2 appears to be suppressed by LPS immune response linked to circadian genes, and PER2 was enhanced, likely due to the use of whole bacteria and not just LPS as in other studies mentioned above. In addition, we expected that TLR4-an important transmembrane immune receptor for LPS and FimH adhesin [50]-would be an important player in bacteria-hepatocytes contact. We thus monitored TLR4 expression initially 24 h after co-culture, and its expression was not rhythmic' neither significantly differed between HepG2 cells in co-culture with E. coli BJ17, BJ23 compared to control without bacteria (see Figure S3). All this information suggests that LPS is probably not a key player in measured variations of circadian clock changes.
The observed amplitude changes of PER1, PER2 and the phase advance of PER3 can be orchestrated by the host innate immune response. Hepatocytes react to bacterial invasion by increasing the levels of pro-inflammatory factors IFN, IL-1β, and TNF-α. If we look closer at how these immune factors affect circadian genes, we can see that IL-1β attenuates PER3 expression and TNF-α downregulates the rhythmic expression of PER1, 2 and 3 [51]. TNF-α does not decrease the expression of BMAL1, but it inhibits the CLOCK/BMAL1 transactivation E-box in the Per and Cry promotors, which results in the decrease in its expression [52]. As an important mediator in the defense against invading pathogens, TNF-α has been induced in hepatocytes exposed to bacteria Salmonella typhimurium and Listeria monocytogenes [53]. TNF-α is probably present also in E. coli co-cultured HepG2 cells, although we were not able to detect it by qPCR due to the low quantity of available RNA.
The reasons for this result might be the short period of circadian sampling (only 24 h) and/or a high percentage of apoptotic HepG2 cells in co-culture with E. coli BJ23 at the start of the circadian sampling ( Figure 4). The chosen E. coli strains were only tested for the most common ExPEC virulence-associated genes [15,54], but our results clearly demonstrate that E. coli BJ23, with more ExPEC virulence-associated genes, causes more HepG2 cells to enter apoptosis or necrosis compared to E. coli BJ17 at time 0 h (Figure 4). After 24 h and 48 h, most of the cells recovered, but a high percentage of necrotic and apoptotic cells occurred after 72 h. After three days, nutrient deprivation and cell density can affect the expression of housekeeping genes [55,56] and the relative expression may appear much higher. On the other hand, after 72 h, the percentage of live cells is at least 60%, and at the same time, instant the relative expression of certain circadian genes in HepG2 cells in co-culture with E. coli BJ23 is 3-5 times lower compared to control cells. It is hard to believe that this situation is a consequence of the high percentage of cell death, only, but more likely a consequence of active immune response. In fact, we measured the relative mRNA expression of IL-6R, receptor of multifunctional cytokine IL-6, which is induced by stimulation of IL-1, TNF-alpha or TLR-4, and expression changed significantly after 72 h ( Figure S2 in the Supplementary Materials).
It was already shown that the circadian gene expression, particularly for the core clock activators CLOCK and BMAL1, is altered in apoptotic cells [57]. Although we cannot conclude that the number of ExPEC virulence-associated bacterial genes is the reason why bacterial strains altered the host's circadian clock differently, our research anyway implies there is a connection that should be investigated further [55,56].
Whether and how individual virulence-associated gene affects the circadian rhythm of the host is still unknown. Knowledge of the ExPEC virulence-associated genes of bacterial strains in microbiota and their connection to the circadian clock could lead to the production of probiotics that would help the host to maintain the homeostasis of the circadian clock in the modern lifestyle (jet lag, shift work, late-night meals, etc.).
In conclusion, this study presents an original system that allows real-time circadian studies of human HepG2 hepatoma cells co-cultured with bacteria. The obtained data on this model revealed that the addition of bacteria retained the rhythmic expression of HepG2 core clock genes except CRY1, and enhanced the circadian behavior of CLOCK and BMAL1. Moreover, individual E. coli strains can have a different impact on the host circadian clock genes.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9040869/s1, Figure S1. Growth characteristics of bacterial strains BJ17 and BJ23, Figure S2: Relative expression of the IL-6R genes as a function of time (24-72 hours), Figure S3: Comparison of circadian behaviour of core clock gene expression between the co-cultures and control, Table S1. Estimation of circadian behaviour of core clock gene expression in HepG2 cells (K) and in co-culture with E. coli strains BJ17 and BJ23, related to Figure 2, Table S2: Relative mRNA expression clock-incubation with no bacteria, control, Table S3: Relative mRNA expression CLOCK-incubation with strain E. coli, strain B17, Table S4: Relative mRNA expression CLOCK-incubation with strain E. coli, strain B23, Table S5: Relative mRNA expression BMAL1-incubation with no bacteria, control, Table S6: Relative mRNA expression BMAL1-incubation with strain E. coli, strain B17, Table S7: Relative mRNA expression BMAL1-incubation with strain E. coli, strain B23, Table S8: Relative mRNA expression PER1-incubation with no bacteria, control, Table S9: Relative mRNA expression PER1-incubation with E. coli, strain BJ17, Table S10: Relative mRNA expression PER1-incubation with E. coli, strain BJ23, Table S11: Relative mRNA expression PER2-incubation with no bacteria, control, Table S12: Relative mRNA expression PER2-incubation with E. coli, strain BJ17, Table S13: Relative mRNA expression PER2-incubation with E. coli, strain BJ23, Table S14: Relative mRNA expression PER3-incubation with no bacteria, control, Table S15: Relative mRNA expression PER3incubation with E. coli, strain BJ17, Table S16: Relative mRNA expression PER3-incubation with E. coli, strain BJ23, Table S17: Relative mRNA expression CRY1-incubation with no bacteria, control, Table S18: Relative mRNA expression CRY1-incubation with E. coli, strain BJ17, Table S19: Relative mRNA expression CRY1-incubation with E. coli, strain BJ23, Table S20: Relative mRNA expression CRY2-incubation with no bacteria, control, Table S21: Relative mRNA expression CRY2-incubation with E. coli, strain BJ17, Table S22: Relative mRNA expression CRY2-incubation with E. coli, strain BJ23, Table S23. Comparison of circadian behaviour of core clock gene expression in HepG2 cells (K) in co-culture with E. coli strains BJ17 and BJ23, related to Figure 2, Table S24. Final dilutions of the bacterial culture in growth curve experiments, related to Bacterial Growth Curves, Table S25. List of used primers for RT-PCR, related to RNA isolation and RT-PCR analysis.