Potential Probiotic Escherichia coli 16 Harboring the Vitreoscilla Hemoglobin Gene Improves Gastrointestinal Tract Colonization and Ameliorates Carbon Tetrachloride Induced Hepatotoxicity in Rats

The present study describes the beneficial effects of potential probiotic E. coli 16 (pUC8:16gfp) expressing Vitreoscilla hemoglobin (vgb) gene, associated with bacterial respiration under microaerobic condition, on gastrointestinal (GI) colonization and its antioxidant activity on carbon tetrachloride (CCl4) induced toxicity in Charles Foster rats. In vitro, catalase activity in E. coli 16 (pUC8:16gfp) was 1.8 times higher compared to E. coli 16 (pUC-gfp) control. In vivo, E. coli 16 (pUC8:16gfp) not only was recovered in the fecal matter after 70 days of oral administration but also retained antibacterial activities, whereas E. coli 16 (pUC-gfp) was not detected. Oral administration of 200 and 500 μL/kg body weight of CCl4 to rats at weekly interval resulted in elevated serum glutamyl pyruvate transaminase (SGPT) and serum glutamyl oxalacetate transaminase (SGOT) levels compared to controls. Rats prefed with E. coli 16 (pUC8:16gfp) demonstrated near to normal levels for SGPT and SGOT, whereas the liver homogenate catalase activity was significantly increased compared to CCl4 treated rats. Thus, pUC8:16gfp plasmid encoding vgb improved the growth and GI tract colonization of E. coli 16. In addition, it also enhanced catalase activity in rats harboring E. coli 16 (pUC8:16gfp), thereby preventing the absorption of CCl4 to GI tract.


Introduction
Human gastrointestinal (GI) tract has a very complex microbiota, with approximately 500-1000 different species [1]. At birth, babies emerge from a sterile environment into one that is loaded with microbes as a result of which the infant's intestine rapidly becomes home to one of the densest populations of bacteria on earth [2]. The endogenous GI microbiota plays a fundamentally important role in health and disease, yet this ecosystem remains to be incompletely characterized [3]. The critical functions of the commensal microbiota include protection against irritable bowel syndrome, inflammatory bowel disease, colorectal cancer, and epithelial cell injury, regulation of host fat storage, and stimulation of intestinal angiogenesis [4,5]. In GI tract, the microbial diversity changes from stomach to rectum. The microbiota of infants possesses three taxonomic groups, whereas healthy adults contain only five phyla and, amongst proteobacteria, E. coli is the predominant commensal microorganism present in the GI tract [2,[6][7][8]. E. coli being a facultative anaerobe colonizes the GI tract at early stages and is proposed to facilitate the colonization of obligate anaerobes belonging to 22 different phyla by the creation of a reduced environment [9]. Many E. coli strains were demonstrated to have probiotic properties [10][11][12][13]. Previously, we isolated several E. coli strains from rat feces demonstrating characteristics such as acid tolerance, antibiotic susceptibility, nonpathogenicity, adherence capability, and antimicrobial activity to the Enterobacteriaceae family [13]. The E. coli 16 showed better adherence and acid tolerance capability along with other characteristics conferring this strain as potential probiotic [13].
Oxygen electron paramagnetic resonance (EPR) imaging technique showed that the GI tract environment fluctuates between anaerobic and microaerobic conditions [14]. In order to adapt the microaerobic environment of GI tract, bacteria downregulate or repress aerobic genes and simultaneously activate anaerobic genes [3]. E. coli being a facultative anaerobe has aerobic respiratory control (ARC) system and fumarate nitrate reductase (FNR) system for aerobic and anaerobic conditions, respectively [15]. Aerobic bacterial respiration is essential for effective competition and colonization of E. coli in microaerobic environment of GI tract [16].
In oxygen poor habitats, Vitreoscilla sp., an obligate aerobe, survives due to efficient oxygen-binding kinetics of Vitreoscilla hemoglobin (VHb) encoded by vgb gene [17]. A heterologous expression of VHb in E. coli resulted in improved cell growth and protein production under microaerobic conditions [15]. Additionally, heterologous expression of vgb in Enterobacter aerogenes reduced H 2 O 2 toxicity [18]. In E. coli, the protective role of VHb is mediated through oxidative stress regulator OxyR, which in turn activates VHb biosynthesis [19,20]. VHb also has been shown to possess peroxidase activity [21,22]. The chimera of superoxide dismutase and VHb protein rapidly detoxified reactive oxygen species (ROS) produced under oxidative stress conditions in E. coli [22].
Carbon tetrachloride (CCl 4 ) causes tissue injury especially in hepatocytes by the formation of reactive trichloromethyl radicals [23]. Trichloromethyl radical reacts with molecular oxygen to form trichloromethylperoxyl radical and oxidizes lipid molecules by hydrogen abstraction especially in hepatocytes. The present study was designed to investigate the effects of E. coli 16 harboring vgb gene on GI tract colonization and CC1 4 induced hepatotoxicity.

Bacterial Strains, Plasmids, and Culture Conditions.
The bacterial strains and the plasmids used in this study are listed in Table 1. E. coli 16 isolate was maintained on Hichrome coliform agar and MacConkey agar plates (HiMedia, Mumbai, India). E. coli DH5 was used for constructing recombinant plasmids. E. coli BL21 was used for expressing the proteins. Luria-Bertani (LB) rich medium [5 g/L yeast extract (HiMedia, Mumbai, India), 10 g/L, Tryptone (HiMedia, Mumbai, India), and 10 g/L NaCl] and M9 minimal medium (12.8 g/L Na 2 HPO 4 ⋅7H 2 O, 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl, 3 mg/L CaCl 2 , and 1 mM MgSO 4 ) were used for plasmid construction and bacterial culture, respectively. NaNO 3 (10 g/L) was added to the medium for induction of the nar promoter and 1 mM FeSO 4 was added as a metal cofactor for VHb protein [18]. Plasmid-containing cells were grown in medium supplemented with 100 g/mL ampicillin. Cloning of the gfp gene into the SmaI site of pUC8:16 results in lacZ-gfp fusion in which gfp is in frame with lacZ sequence. The recombinant plasmid was confirmed by restriction digestions. The plasmids pUC-gfp and pUC8:16gfp were independently transformed in the potential probiotic E. coli 16 using the CaCl 2 method [24]. The transformants were screened by their fluorescence at 365 nm in ultraviolet transilluminator.

Preparation of E. coli 16 Culture and Cell-Free Extracts
for Catalase Assays. Luria broth culture was grown as 60 mL of culture in a 100 mL flask. The cells were incubated at 37 ∘ C using agitation rates of 75 rpm and were treated with CCl 4 (65 mM) at 0.4 to 0.5 O.D., that is, midlog phase, and incubated for 30 h. The cells were then harvested by centrifugation at 9,200 g for 2 min at 4 ∘ C. The cell pellet was washed once with 50 mM phosphate buffer (pH 7.0) followed by resuspension in the same buffer. The cells were subjected to sonication (Branson Sonifier Model 450) for a total period of 1 min at a pulse rate of 15 s in an ice bath, followed by centrifugation at 9,200 g at 4 ∘ C for 30 min to remove the cell debris. The supernatant thus obtained was used as cell-free extract for the catalase assay.

Catalase Assay of Cell-Free Extract.
The cell-free extract was added in a cuvette followed by addition of 30 mM H 2 O 2 prepared in 50 mM potassium phosphate buffer (pH 7.0). The decrease in absorbance was measured at 240 nm for 1 min to determine the catalase activity [25]. The molar extinction coefficient of 43.6 M/cm was used to determine the catalase activity and the activity was reported as units/min/mg of protein.

Animal Experiments.
Male Charles Foster rats were housed in the departmental animal house facility under controlled room temperature (21 ± 2 ∘ C). The animals were fed chow diet and water ad libitum. The experiments were carried out after the approval of the Animal Ethical Committee of Department of Biochemistry, The MS University of Baroda, Vadodara (Approval no. 938/A/06/-CPCSEA). The guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) were followed.

GI Tract Colonization Experiments.
Three-month aged rats were given drinking water containing streptomycin sulphate (5 g/liter) for 24 h to remove the existing resident facultative microflora and then starved for food and water for 18-20 h. The rats were divided into two groups and were fed approximately 10 9 CFU of E. coli 16 (pUC-gfp) and E. coli 16 (pUC8:16gfp), respectively, in 1 mL of 20% sucrose once a day for up to 3 days. After the bacterial suspension was ingested, food and water were restored, and fecal plate counts were determined at regular intervals till the 70th day. Fecal samples were homogenized, serially diluted in 0.85% saline, and plated on Luria agar plates containing ampicillin (100 g/mL). After 24 h, the plates were inspected under UV light for the fluorescence. As soon as the reduction of fluorescent colonies of fecal samples was noted, rats were given ampicillin (50 mg/kg body weight) (days 23-25 and days 48-51) in drinking water [16,26]. Selected fluorescent colonies from fecal samples were screened for vgb gene. The colony PCR was carried out on using a set of specific primers that anneal to a region of the vgb gene in E. coli 16 (pUC8:16gfp) and amplify a 714 bp fragment. Similarly, least dilution of fecal homogenate was plated on Luria agar plates containing lawn of E. coli DH5 to show the inherent antimicrobial property of E. coli 16 (pUC8:16gfp). The pUC8:16gfp is a nonmobilizable plasmid and does not get transferred horizontally [26].

Effect of E. coli 16 (pUC8:16gfp) under Oxidative Stress.
A total of 15 rats (14 to 16 months) were equally divided into five groups ( = 3). Group I served as normal control and was orally given saline for 3 days followed by 2 weeks interval up to 45 days. Group II served as potential probiotic E. coli 16 (pUC8:16gfp) where rats were fed the culture orally with saline for 3 days followed by 2 weeks interval up to 45 days. Group III served as normal control with CCl 4 and was orally given saline for 3 days followed by 2 weeks interval up to 45 days. Further, two doses (200 L and 500 L) of CCl 4 were given with olive oil as carriers at weekly interval and the antioxidant parameters in plasma and liver were monitored to assess the liver function. Group IV (potential probiotic E. coli 16 (pUC-gfp) with CCl 4 ) served as vector control and the same procedure was followed as that of Group III. Group V [potential probiotic E. coli 16 (pUC8:16gfp)] with CCl 4 served as test and the same procedure was followed as that of Group III. At the end of the 2nd dose, on the 3rd day, rats were mildly anesthetized and blood was collected via retroorbital sinus followed by plasma separation for further biochemical analysis. Later, animals were sacrificed by decapitation under mild anesthesia and liver was excised and stored at −80 ∘ C for further estimations.

Assessment of Liver Function.
Liver damage in the above mentioned groups of rats was assessed by estimating the plasma levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) using commercially available mono single test IFCC, kinetic SGOT and SGPT test kits from Reckon Diagnostics Pvt. Ltd, Vadodara, Gujarat, India, as per the manufacturer's instructions.

Estimation of Lipid Peroxidation and Catalase Activity.
Liver samples (100 mg/mL) were homogenized in 50 mM potassium phosphate buffer and centrifuged at 10,000 rpm for 15 min. The supernatant thus obtained was used for estimating the lipid peroxidation levels and catalase activity. Catalase activity was expressed as enzyme activity per mg of protein. The protein concentration in each fraction was determined by modified Lowry method [27], using bovine serum albumin as standard. The mean malondialdehyde (MDA) content ( mol/mg protein) was used as an indicator of lipid peroxidation and assayed in the form of thiobarbituric acid-reacting substances (TABRS) [28]. Catalase activity was measured using the method described previously [25].

Microscopic Examination of Liver.
Liver samples were fixed using 4% buffered paraformaldehyde followed by dehydration in graded alcohol series and embedded in paraffin wax. About 4-5 m thick sections were cut (by Leica RM 2155 Microtome) followed by staining with hematoxylin and eosin for examination using Leica microscope.

Statistical Analysis.
Statistical evaluation of the data was performed by one way analysis of variance (ANOVA) followed by Bonferroni's corrections for multiple comparisons. The results were expressed as mean ± SEM using GraphPad Prism version 5.0 for Windows, GraphPad Software, San Diego, California, USA.

Effect of vgb Gene Expression on E. coli 16 (pUC8:16gfp).
A significant effect of vgb expression was observed as E. coli 16 (pUC8:16gfp) demonstrated an increase in growth rate under microaerobic condition compared to its pUC-gfp vector control (Figure 1).

vgb Gene Expression Enhances In Vitro Catalase
Activity of E. coli 16 (pUC8:16gfp). In the presence of CCl 4 , catalase activity of E. coli 16 (pUC8:16gfp) was increased by 1.8-fold as compared to E. coli 16 (pUC8-gfp) vector control ( Figure 2). This suggests that vgb gene was expressed under microaerobic environment and was functional in E. coli 16 (pUC8:16gfp).  (Figure 3(a)). Thus, the retention time of potential probiotic E. coli 16 (pUC8:16gfp) was significantly improved in the GI tract of rats. On the 48th day of postfeeding (Figures 3(b) and 3(c)), ampicillin resistant bacterial colonies were detected from fecal samples. These colonies demonstrated fluorescence as well as antimicrobial properties suggesting that both properties were retained in the E. coli 16 (pUC8:16gfp).

SGOT and SGPT Activity in Plasma.
Oral administration of CCl 4 to Groups III (normal control with CCl 4 ) and IV (vector control) rats resulted in significantly elevated ( < 0.001) serum levels of SGPT and SGOT as compared to Group I (control) and Group II [prefed with potential probiotic E. coli 16 (pUC8:16gfp)] untreated rats. Treatment of CCl 4 to Group V rats [prefed with potential probiotic E. coli 16 (pUC8:16gfp)] resulted in significantly decreased ( < 0.05) activities of SGOT and SGPT enzymes as compared to Group III and Group IV rats (Figures 4(a) and 4(b)). Rats treated with CCl 4 and prefed with potential probiotic E. coli 16 (pUC8:16gfp) resulted in near to normal plasma SGPT and SGOT levels.

Liver Lipid Peroxidation Level and Catalase Activity.
Catalase activity was significantly decreased in the liver homogenate of CCl 4 treated Group III and Group IV rats as compared to control groups. Potential probiotic E. coli 16 (pUC8:16gfp) Group V rats showed significantly ( < 0.05) increased catalase activity compared to CCl 4 treated Group III rats (Figure 4(c)). Slight decrease in the mean MDA levels was found in the liver of Group V (CCl 4 -exposed) rats compared to Group III rats (Figure 4(d)).

Histopathological Analysis of Liver.
Histopathological analysis of liver cells using hematoxylin and eosin stains in Group III and Group IV rats treated with CCl 4 revealed extensive liver damage, characterized by the disruption of the lattice nature of the hepatocytes, damaged cell membranes, degenerated nuclei, disintegrated central veins, and damaged hepatic sinusoids as compared to the liver of Groups I and II (control) rats. However, in Group V rats [treated with CCl 4 and prefed with potential probiotic E. coli 16 (pUC8:16gfp)], only minimal disruption of the hepatic cellular structure was observed ( Figure 5).

Discussion
Probiotic bacteria exert their effects by competing with potentially pathogenic bacteria for ecological niches, thereby preventing their colonization. The exact mechanism of colonization of E. coli in the GI tract is not clear, but it is known that the respiration of E. coli in GI tract is very much essential for its successful colonization and competitiveness in the GI tract [16]. Colonization and competitiveness of facultative anaerobes, that is, E. coli, depend on their respiratory flexibility which in turn depends on high-affinity cytochrome bd oxidase. Previous study has shown that VHb improves the oxygen uptake rate of E. coli under microaerobic condition, by 5-fold and 1.5 increase of cytochrome bo 3 and cytochrome bd oxidase, respectively [29]. In the study, VHb expression was enhanced by low oxygen tension via a fumarate and nitrate reductase regulator-(FNR-) dependent mechanism [29]. When a vgb gene is introduced into E. coli, the cell growth and yield of the target protein were increased significantly [16,18,[30][31][32][33][34]. In the present study, E. coli 16 (pUC8:16gfp) plasmid expressing vgb gene was also found to increase the growth rate in microaerobic condition as well as improve the GI tract colonization and enhance the catalase activity. The expression of vgb gene significantly improved the colonization of potential probiotic E. coli 16 (pUC8:16gfp) in rat GI tract, possibly due to improved cell growth and better respiratory adaptation under low oxygen tension. Superoxide radicals (O 2 − ) formed within biological systems are toxic to living cells. Trichloroperoxyl radical (CC1 3 OO • ) synthesized from (O 2 − ) and CC1 4 has a highly toxic effect on metabolic oxidizing activities presumably because of the electron-withdrawing nature of the trichloromethyl group [23,35]. Heterologous expression of nonheme catalase in Lactobacillus lactis improved the antioxidant status and alleviated the risk of 1,2-dimethyl hydrazine induced colon cancer [36,37]. Near to normal levels of SGPT and SGOT activity in CCl 4 treated rats with E. coli 16 (pUC8:16gfp) plasmid demonstrate the protection of the toxic effects of CCl 4 in the liver. Previous reports have suggested that the protective effects could be attributed to the peroxidase activity of VHb [21,38]. Additionally, VHb is known to decrease the oxidative stress caused by H 2 O 2 by enhancing the catalase activity [18]. The present study also found 1.8-fold increased activity of catalase contributed by vgb gene under in vitro condition. It has been shown that VHb in E. coli induces the expression of catalase-peroxidase G (katG) and superoxide dismutase A (sodA) genes, thereby protecting from damage caused by ROS [19]. In comparison, when vgb gene was expressed in E. coli oxyR mutant, the vgb expression was increased but the strain showed high sensitivity to oxidative stress without induction of antioxidant genes. Thus, oxidative stress regulator OxyR mediates the protective effect of vgb under oxidative stress [19].

Conclusion
The present investigation showed that vgb gene when expressed in a potential probiotic E. coli 16 strain increased its GI tract colonization, thereby improving its survival. In addition, vgb gene being an antioxidant, it detoxified the CCl 4 in GI tract and thereby reduced the hepatotoxicity in rats. Hence, the retention of probiotics in GI tract is thus enhanced; it reduces the doses to maintain an effective probiotic count in the GI tract. These additional benefits may increase the efficiency of the probiotics making them more effective in minimum dose intervals.