Colicin E2 Expression in Lactobacillus brevis DT24, A Vaginal Probiotic Isolate, against Uropathogenic Escherichia coli

Novel therapeutic approaches are needed to combat the urinary tract infection in women. During menstruation elevated protein concentration and increase in oxygen and carbon dioxide concentrations with decrease in vaginal Lactobacilli all together contribute to urinary tract infections. Lactobacillus species are a predominant member of the vaginal microflora and are critical in the prevention of a number of urogenital diseases. In order to increase antimicrobial potential of vaginal Lactobacilli, bacteriocin colicin E2 which has specific activity against uropathogenic Escherichia coli has been overexpressed in vaginal probiotic Lactobacillus brevis DT24. Recombinant Lactobacillus brevis DT24 expressing colicin E2 showed much higher inhibitory activity against uropathogenic Escherichia coli than wild type L. brevis DT24 in vitro. Efficacy of probiotic Lactobacillus brevis DT24 expressing colicin E2 protein is required for further in vivo evaluation.


Introduction
Urinary tract infection (UTI) is the most widespread infection in women worldwide after intestinal infection [1]. UTIs affect an estimated 1 out of 3 women before the age of 24 [2,3]. Up to 40 to 50% of the female population will develop a symptomatic UTI at some time during their lives [2,3] or develop complicated UTIs [4]. Recurrent UTI (rUTI) is a common syndrome in young healthy women. Previous studies suggest that 27% to 44% of women, who experienced an initial UTI, develop rUTI [5,6].
UTI has the potential for severe and life-threatening sequelae if left untreated or undertreated. Possible sequelae include pyelonephritis which can lead to renal scarring and sepsis [7]. UTI can be particularly dangerous in pregnant women in whom it has been shown that up to 50% of those with asymptomatic bacteriuria (ABU) leads to develop pyelonephritis. In addition, these women experience higher rates of intrauterine growth restriction and low birth weight infants. The presence of a UTI has also been shown to increase the risk of preterm labor, preterm birth, pregnancy-induced hypertension, preeclampsia, amnionitis, and anemia [8].
Colicin E2 (ColE2) is a proteinaceous bacterial toxin produced by some strains of Escherichia coli that exhibits inhibitory activity against uropathogenic E. coli [22] via binding to an outer membrane receptor-the TonB-dependent vitamin B12 transporter, BtuB [23].

ISRN Urology
Earlier reports suggest that there is an increased antibiotic resistance in E. coli. Initially, resistance was limited to certain specific antibiotics, such as ampicillin or trimethoprim [24], but recently the horizon of resistance has expanded, with the emergence of broad resistance to third generation antibiotics [25][26][27][28].
Lactobacillus species are an important group of bacteria that inhabit the gastrointestinal tract and represent the predominant microorganism found in the healthy vaginal ecosystem [29][30][31][32][33], producing a variety of compounds [34][35][36][37][38] that inhibit potentially pathogenic microorganisms. It is for this reason that Lactobacillus species have been studied as a potential probiotic for the prevention and treatment of urogenital disease in women [39][40][41][42]. During menstruation, the vaginal pH becomes neutral, most likely due to the influx of menses blood, which has a pH range of 6.9 to 7.2 [43]. In addition, menses blood in the vagina also results in an elevated protein concentration [43] and increase in oxygen and carbon dioxide concentrations [44], leading to decrease in vaginal Lactobacilli, which all contribute to colonization of uropathogenic E. coli which leads to UTIs [45][46][47][48].
As a means of increasing the antimicrobial capabilities of Lactobacillus species against uropathogenic E. coli (UPEC), probiotic L. brevis DT24, isolated from vagina of healthy women [49], was used for overexpression of colicin E2.
The objective of this study was to express ColE2 structural gene ceaB and immunity gene ceiB from E. coli into probiotic Lactobacillus brevis DT24, secreting colicin E2 to exclude a uropathogenic E. coli competitively and completely. In this study genetically engineered probiotic Lactobacillus brevis DT24 would exert its antimicrobial effect against the target pathogen directly through the expression of ColE2 gene and indirectly through beneficial properties inherent in probiotics.

Bacterial Strains, Plasmids, and Growth
Conditions. Bacterial strains used in this study are listed in Table 1. Lactobacillus brevis DT24 (NCBI Accession no. JX163909) was routinely cultured in MRS broth (HiMedia, Mumbai) at 37 ∘ C for 48 h. For the analysis of expression of colicin E2, recombinant strains were grown in basal MRS medium supplemented with 2% xylose. Escherichia coli DH5 , Escherichia coli BL21 D3, and Escherichia coli NCTC 50133 were routinely cultured in Luria Bertani (LB) broth and agar at 37 ∘ C for 16 to 24 h and uropathogenic Escherichia coli MTCC 729 (Microbial Type Culture Collection, Chandigarh) was routinely cultured in nutrient broth and agar at 37 ∘ C for 16 to 24 h. Chloramphenicol (Cm) and streptomycin (Stpr) were used for the selection of plasmids.

DNA Manipulation, E. coli Competent Cell Preparation,
Transformation, and PCR. Plasmid DNA was isolated using GeneJet Plasmid Miniprep Kit as per instruction (Fermentas). DNA cloning and transformation procedures followed as previously described [51]. Restriction enzymes were purchased from New England Biolabs. Ligation was carried out by using Rapid Ligation Kit (Fermentas).  [51]. Colicin E2 gene fragment of about 2.01 kb encoding the colicin E2 structural gene (ceaB) and colicin E2 immunity gene ceiB was obtained from the plasmid pColE2-P9 (E. coli genetic resource centre, Yale University, USA) by polymerase chain reaction (PCR) amplification with the primers 5 -GGATCCATGAGCGGTGGCGAT-3 (forward) containing a BamHI site (underlined) and 5 -CTCGAGTCAGCC-CTTTTAAATCCTGA-3 (reverse) containing an XhoI site (underlined). PCR conditions were as follows: 30 cycles of 30 s at 94 ∘ C, 30 s at 58 ∘ C, and 2 min 20 s at 72 ∘ C after denaturing for 4 min at 94 ∘ C.
The PCR product of colicin E2 gene was cleaved with BamHI and XhoI restriction endonuclease and inserted into the corresponding sites of pPSL111.3 digested by BamHI and XhoI, respectively, giving rise to pSLP-ColE2 ( Figure 1).
Electroporation of L. brevis DT24 was carried out as previously described [52] with some modifications. In brief, a 2% inoculum of an overnight culture was grown in MRS medium supplemented with 1% glycine at 37 ∘ C until the OD660 of culture was 0.

Molecular Weight Determination.
Transformed ColE2 gene having Lactobacillus brevis DT24 was grown in MRS medium at 37 ∘ C for 24 hr and centrifuged (10,000 rpm, 30 min, and 4 ∘ C) to collect supernatants. The collected supernatants were filter sterilized (0.20 m; Axiva). Ammonium sulfate was slowly added to the cell-free supernatants to 60% saturation and stirred for 4 h at 4 ∘ C and centrifuged (10,000 rpm, 30 min, and 4 ∘ C). The precipitate was resuspended in 10 mL of 25 mM ammonium acetate buffer (pH 6.5) and desalted by dialysis using a 1,000 Da cutoff dialysis membrane (Sigma) against the same buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for further separation, as described by Laemmli [53].

Antimicrobial Activity of Recombinant L. brevis DT24
Expressing Colicin E2. Antimicrobial activity of cell-free supernatant (filter sterilized) was measured by well diffusion assay. Filtrates were neutralized (set to pH 6.5) with 5 N NaOH. Nutrient agar plates were flooded with pathogenic bacteria (0.1% of overnight grown uropathogenic E. coli strain), air-dried, and then 6 mm diameter wells were punctured in each plate. The prepared supernatants were poured into respective wells (25 L) and incubated for 24 h at 37 ∘ C. L. brevis DT24 was used as negative control and E. coli NCTC 50133 was used as positive control.

Construction of an Expression
Vector. Plasmid pSLP111.1 is a high-copy-number plasmid which includes a constitutive XylA promoter, secretion signal SlpA, cell wall anchoring domain of PrtP, and the chloramphenicol resistance gene (cat gene). Plasmid pSL-ColE2 was constructed by inserting a 2.01 kb PCR amplicon obtained from pColE2-P9 DNA containing ceaB and ceiB into pSLP111.1 (Figure 1). Transformants containing pSL-ColE2 demonstrated inhibitory activity against the uropathogenic E. coli (Figure 1). The presence and orientation of the ceaB and ceiB insertion in pSL-ColE2 was verified by PCR ( Figure 2). incubated with vigorous shaking (200 rpm), and cell growth was monitored by checking optical density at 600 nm. After 24 hrs of growth 52 kDa protein was observed in SDS-PAGE analysis (Figure 3).

Antimicrobial Activity of Recombinant L. brevis DT24
Expressing Colicin E2. Antimicrobial properties of transformed L. brevis DT24-ColE2 showed higher zone of inhibition (56 mm) compared to Wild Type L. brevis DT24 (23 mm) were shown in Figure 4. But there is no difference in the inhibition zone showed by L. brevis DT24 ColE2 and E. coli NCTC 50133.

Discussion
Various techniques have identified Lactobacillus species as the predominant microorganism found in the vaginas of most healthy and fertile women [40,41]. Lactobacillus species have been studied as a potential probiotic for the prevention and treatment of urogenital disease in women [39][40][41][42]. During menstruation, the vaginal pH becomes neutral, most likely due to the influx of menses blood, which has a pH range of 6.9 to 7.2 which leads to lower Lactobacilli number in vagina and chances of infections like urinary tract infections and bacterial vaginosis. Colicin E2 production by a nonpathogenic organism may have clinical applicability as a means to prevent catheterassociated urinary tract infection [22]. Evans et al. [54] demonstrated the possibility of developing oral whole-cell vaccines against diarrhea caused by enterotoxigenic E.coli  by modifying this E. coli by the in situ destruction of chromosomal and plasmid DNAs by ColE2. The colicin operon is carried on a plasmid and includes a structural gene (ceaB) encoding for the bacteriocin, an immunity gene (ceiB) that protects the producer cell from the toxin, and a lysis gene (celB) that leads to death of the producer cell and release of ColE2 into the surrounding medium [23]. Sensitivity of gram-negative microorganisms to ColE2 is conferred by the binding of the bacteriocin to an outer membrane receptor, the TonB-dependent vitamin B12 transporter, BtuB [23]. After transport across the membrane, ColE2 acts as an endonuclease, degrading the DNA of the sensitive cell. During UTIs uropathogenic E. coli overexpresses surface protein BtuB which can act as receptor for binding colicin E2.
The colicin E2 gene (ColE2) from E. coli cloned in shuttle secretion vector (pSLP111.3) and successfully expressed in our lab probiotic isolate Lactobacillus brevis DT24 and its impact on the inhibitory activity of host organism were examined. The expression of ColE2 in Lactobacillus brevis DT24 was studied by isolating proteins extracellularly.
One of the challenges of transforming ColE2 in Lactobacillus is the differences in the transport mechanisms of bacteriocins in gram-negative and gram-positive microorganisms [55]. In gram-negative microorganisms, ColE2 is thought to be released into the surrounding medium after CelB-mediated lysis of the producer cell. Expression of celB leads to changes in the cell envelope and results in activation of Omp LA, an outer membrane phospholipase A [23]. Mutation or deletion of the lysis protein has been shown to interfere with release, and in such cases, colicin remains in the cytoplasm [23]. In gram-positive microorganisms, secretion does not occur through cell lysis and is not a lethal event for the cell. Instead, secretion is dependent on a signal peptide, which typically contains conserved double-glycine regions and is mediated by a bacteriocin-specific transport system or the sec-dependent export pathway [56]. Although ColE2 does not contain a signal peptide to direct the secretion of the protein, it is predicted to contain 6 double-glycine regions at the N terminus, which may function in a manner analogous to a signal peptide. Thus, ColE2 may be secreted by a grampositive host without lysis of the producer cell. This feature is important if the genes encoding for colicin production are to be transferred to and expressed by a Lactobacillus [57].
This study demonstrated that genes associated with bacteriocin production from a gram-negative microorganism could be cloned, expressed, and secreted by a gram-positive microorganism in the absence of a lysis protein (CelB) and with addition of a signal peptide. In the present work, genes associated with ColE2 production (ceaB and ceiB) were transferred to L. brevis DT24, probiotic isolate from vagina. The level of ColE2 production by the colicin-producing transformants of L. brevis DT24 was similar to that of E. coli NCTC 50133, from which the ColE2-encoding genes (pColE2-P9) were derived.
Secretion of ColE2 proteins into the surrounding medium by E. coli NCTC 50133 and the pSLP111.3-ColE2 transformants occurred before cell leakage was observed. The mechanism proposed for secretion of ColE2 from E. coli involves release of the colicin caused by the lysis protein CelB [58][59][60][61][62][63]. Braun et al. [64] found that inactivation of celB resulted in decreased release of colicin from the cells, compared with cells containing intact celB.
Cloning and expression of ColE2 in L. brevis DT24 allowed evaluation of the transformant as a bioactive compound for use in treatment of UTI. Similar strategies were used for treatment of Staphylococcus aureus infection by expressing antimicrobial protein lysostaphin in vaginal probiotic Lactobacillus plantarum WCFS1 [65] and inhibition of HIV by expressing anti-HIV proteins which were capable of blocking the HIV entry into human peripheral blood mononuclear cells in probiotic organism Lactobacillus reuteri RC-14 [66].

Conclusion
This study has demonstrated that the expression of E. coli colicin E2 (ColE2) into Lactobacillus showed increased expression of colicin E2 in extracellular level to inhibit the infectious disease occurred by uropathogenic E. coli. The probiotic properties of host Lactobacillus brevis DT24 were increased in the terms of antimicrobial activity against pathogenic E. coli. Oral administration of probiotics has clear effects on the numbers and activities of intestinal and fecal bacteria. The administration oral probiotics help to reduce the transfer of yeast and urogenital pathogenic bacteria from the rectum to vagina [67]. It may be possible to use these L. brevis DT24-ColE2 probiotics in biotherapy (i.e., as vehicles for the secretion of colicin E2 in the gastrointestinal tracts as well as urovaginal tract for the treatment of UTI as well as other gastrointestinal infectious diseases).

Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.