Construction and development of FimH lectin domain for rising immune response after injection by uropathogenic E. coli

Abstract

Uropathogenic E. coli is one of the major agents of urinary tract infection. Today, no effective treatment or vaccine against this infection is exist. Accordingly, in the present study, a genetic constrruct for inducing of cellular immune system was designed. At first, fimH gene from E. coli 35218 was amplified using PCR. PCR product inserted into pET23a expression vector and the recombinant vector was analysed by sequencing. The vector was transformed to E. coli strain Origami and the protein was expressed under the 1 mM IPTG. FimH was purified with Ni-NTA column and the purified protein was used for immunization of BALB/c. Two weeks after the last injection, lymphocyte proliferation assay was carried out. In addition, IL-4 and IFN- $\gamma$ cytokines, total antibody serum, IgG1 and IgG2a isotypes were quantified. Finally, protection ability of the vaccine in bladder and kidney infection of mice was evaluated.

The results indicated that cellular immune response has a main protective role against UTI and FimH, as a vaccine candidate, significantly increase lymphocyte proliferation, IFN- $\gamma$ response and total antibody amount. Immunization of mice with FimH conferred effective protection of kidney and bladder against urinary tract infection by uropathogenic E. coli ( $P<$ 0.002). It can be concluded that, the current FimH will be valuable for more trying to prepare a new vaccine against UTI.

Keywords

Urinary tract infection uropathogenic Escherichia coli fimh mouse model

1. Introduction

Urinary tract infection (UTI) is the widespread infection in young women which affect urinary system (kidneys, ureters, bladder and urethra) [1, 2]. Uropatho-genic E. coli (UPEC) is the main cause of community-acquired UTIs, which regularly persists in feces [3, 4]. The UTI is usually self-limiting, but UPEC establishing a persistent infection via penetrate to bladder cells and intracellular amplification [5]. Likewise, acute pyelonephritis is assumed to be the result of an ascending infection from the bladder to the kidney. Some protection mechanisms from microbial colonization were made urinary tract to be sterile like urinary flow, soluble factors that are secreted into urine, anatomical barriers such as the glycoprotein plaque and a layer of mucus [6, 7, 8]. However, pathogenic E. coli bacteria have several virulence factors such as adhesion molecules and toxins which can cause acute infections [8].

Antibiotic treatment, is generally effective for elimination of the infecting strain. However, increase in multidrug resistance, allergic reaction to certain drugs and failure to prevent recurrent infections represent significant barriers to treatment [9]. As a result, UTI prevention by immunization using virulent antigens of bacteria represents a gap that must be addressed. While several clinical trials of vaccine candidates have currently been tested against urinary tract infection in human [10, 11, 12, 13], no suitable results have been found so far. Therefore, targeting critical molecules such as FimH as a vaccine candidate can lead to an increase in vaccines efficacy [13]. Immunization with UPEC antigens like FimH stimulates increases in urinary and serum antibody titers that correlate with reductions in bladder and/or kidney bacterial load [14].

The bacterial adhesion FimH is a receptor-recogniti-on element and virulence factor located on type 1 pili of UPEC, and plays an integral role in the pathogenesis of UPEC in urinary tract infection (UTI) and Crohn’s Disease (CD). This protein is produced as a precursor of 300 amino acids which is processed into a mature form of 279 amino acids and is folded into two domains, an N-terminal lectin domain (residues 1–156) linked by a short tetrapeptide loop to a C-terminal organelle integration domain (residues 160–279) [15, 16, 17].

In the present study, a FimH lectin domain conjugated to His-tag was cloned, expressed, purified and injected to animal model. Since FimH is not soluble, here a construct consisting of the FimH binding domain fused to His-Tag to increase the antigen solubility was constructed. Consequently, immune response against this protein was evaluated in the cellular and murine model. Here we show that this construct is immunogen, soluble and easy to purify.

2. Materials and methods

2.1 Amplification of E. colifimH gene

The fimH gene used as a basis for manipulations was originally cloned from the E. coli 35218 (purchased from Pasteur Institute, Karaj, Iran). DNA was extracted using the AccuPrep ${}^{\text{\textregistered}}$ Genomic DNA Extraction Kit (Bioneer) from E. coli 35218 and amplified using PCR. Primers were designed based on the fimH gene of UTI89 strain (GenBank accession nos. NC_007946.1). PCR reactions were performed by Eppendorf thermocycler, in 50 $\mu$ l volume containing 2 $\mu$ l of DNA template, 5 $\mu$ l of 10x reaction buffer, 2 $\mu$ l of dNTPs (10 mM), 2 $\mu$ l of MgCl ${}_{2}$ (50 mM), 10 $\mu$ l of each primer (20 pmol, FW (5 ${}^{\prime}$ -T C C C G T T A C A G G T C A G A G C-3 ${}^{\prime}$ ) and RW (5 ${}^{\prime}$ -G T G C A G G T T T T A G C T T C A G G-3 ${}^{\prime}$ )). Run conditions was carried out by pfu DNA polymerase as follows: 94 ${}^{\circ}$ C for 4 min; 35 cycles at 94 ${}^{\circ}$ C for 1 min, 35 cycles at 57 ${}^{\circ}$ C for 1 min, 35 cycles at 72 ${}^{\circ}$ C for 1 min; and then a final step at 72 ${}^{\circ}$ C for 5 min. The whole PCR mixture was run on a 1% agarose gel, and the resulting band was excised and purified.

The PCR products of fimH gene was ligated into pBluescriptIISK cloning vector by EcoRV restriction enzyme. T4 DNA ligase (Fermentas) was used for the ligation. The resulting plasmids were transformed into competent E. coli (Top10F’) (Stratagene) by chemical method (CaCl ${}_{2}$ ). The MacConkey plates containing 100 $\mu$ g/ $\mu$ l ampicillin and 34 $\mu$ g/ $\mu$ l tetracycline were used for selection of transformed colonies. The white colonies were selected and following an overnight cultivation, were subjected to plasmid extraction and PCR.

2.2 Expression of the fimH genes

The fimH gene cloned in pBluescript II SK (Stratagene) vector were used as the source of DNA for cloning into expression vector pET23a (Novagen). After amplification, the PCR products were gel-purified and digested with the EcoRI and HindIII restriction enzymes. The digested products were cloned into the NdeI and EcoRI sites of expression vector pET28a under the T7 promotor with histidine Tag (his6) to generate proteins with His6 at the C-terminal of the protein. The plasmid contains: the NdeI restriction site (ATG used as start codon) fused to fimH lectin domain sequence and six histidine tag ligated into the stop linker (GAATTC) and EcoRI restriction site. The ligated plasmid was transformed into competent E. coli origami (DE3). The selected clones were analyzed by gel electrophoresis, PCR and sequencing. Recombinant E. coli origami (DE3) cells were grown overnight in Luria broth (LB) medium containing kanamycin (50 $\mu$ g/ml) at 37 ${}^{\circ}$ C. Then, expression of the cloned gene was induced by 1 mM IPTG.

2.3 SDS-PAGE and western blot

For analysis of expression of proteins, the 15% SDS-PAGE was used. For western blot, the samples were separated by SDS-PAGE and transferred in nitrocellulose membrane using a liquid transfer system (Bio-Rad). His-tag (invitrogen) and HRP conjugated antibodies were use as primary and secondary antibodies, respectively.

2.4 Purification of the fimH-his protein

E. coli origami (DE3) cells transformed with the plasmid coding for FimH lectin domain-His were grown to an OD ${}_{600}$ of 0.6 and FimH lectin domain-His production was induced with 1 mM IPTG for 1 h. Bacterial cells were harvested by centrifugation and resuspended in a buffer containing 20 mM Tri-HCl, 0.5 M NaCl, 0.3% Triton-X-100, 10 mM Imidazole pH 8.0 and the solution incubated on ice with gentle agitation for 10 min. The cells were harvested again by centrifugation and resuspended in the same volume of ice-cold 5 mM MgSO ${}_{4}$ . Following a 10 min incubation on ice the cells were centrifuged again and the supernatant (cold osmotic shock fluid) was dialyzed extensively against a buffer containing 50 mM Na-phosphate pH 7.8, 300 mM NaCl. This resulted in essentially a preparation of periplasmic proteins in PBS. The His-tagged FimH lectin domain protein was purified from this periplasmic fraction by Ni-NTA affinity chromatography according to standard procedures (Qiagen). Samples were subjected to SDS-PAGE (15%) gel electrophoresis and confirmed using western blot techniques

2.5 Mice and immunization experiments

Six to eight week inbred female BALB/c mice were purchased from Pasteur Institute of Iran (Karaj, Iran). Mice were housed for 1 week before the immunization. The study was conducted following the approval of the institutional animal ethics committee. The subjects were kept in the animal house in a room temperature of 22 $\pm$ 3 ${}^{\circ}$ C under a 12:12 h light-dark cycle; they also had free access to food and water and were allowed to adapt to the laboratory conditions for at least one week prior to the first day of test. The endotoxin level of extracted FimH was detected by the limulous amoebocyte lysate test (LAL test) according to the manufacturer’s standard protocol (Thermo scientific, USA). Experimental mice ( $n=$ 18) were divided into 3 groups ( $n=$ 6). The groups 1 to 3 were intramuscular injected two times with 2 week interval with 100 $\mu$ g of FimH adsorbed on 2 mg alum, or 100 $\mu$ g of FimH emulsified in 0.2 ml complete freund’s adjuvant. PBS injections were considered as control group.

2.6 Lymphocyte proliferation assay

Two weeks after final immunization, the spleens of mice were dissected and resuspended in cold PBS containing 5% FBS. RBCs were lysed with lysis buffer and, after centrifugation, the pellet was resuspended and adjusted to 3 $\times$ 10 ${}^{6}$ cells/ml in RPMI 1640 (Gibco, Germany) supplemented with 10% FBS, 4 mM/L-glutamine, 1 mM sodium pyruvate, 50 $\mu$ m 2ME, 100 $\mu$ g/ml streptomycin and 100 IU/ml penicillin. Then, 100 $\mu$ l (2 $\times$ 10 ${}^{5}$ cells/well) of the cell suspension was dispensed into each well of 96-well culture plates in triplicate and stimulated with 10 $\mu$ g/ml of the FimH protein and incubated at 37 ${}^{\circ}$ C in a 5% CO ${}_{2}$ incubator for 72 h. Phytohemagglutinin-A (5 $\mu$ g/ml, Gibco), unstimulated wells and complete culture medium were used as a positive control, negative controls and a blank, respectively. After 72 h, 100 $\mu$ l of 5-bromo-2-deoxyuridine labeling solution was added to each well and incubated for 18 h. Then, 100 $\mu$ l of the peroxidase-conjugated anti-BrdU antibody was added to each well for 2 h, the plates were washed five times with PBS, 100 $\mu$ l of the TMB substrate was added to each well in the dark condition and the reaction was stopped by adding 100 $\mu$ l of 2 M H ${}_{2}$ SO ${}_{4}$ . The optical density of each sample (OD ${}_{450/69}$ 0) was measured at a wavelength of 450 nm with background subtraction at 690 nm by using an ELISA plate reader. Stimulation Index (SI) was calculated according to the following equation: SI $=$ (OD ${}_{450}-$ OD ${}_{690}$ of antigen-treated cells)/(OD ${}_{450}-$ OD ${}_{690}$ of untreated cells).

2.7 Total antibody ELISA and determination of igG1, igG2a subclasses

Sera of experimental mice were collected two week after last injection (third) and the serum IgG antibody responses were measured by ELISA. Briefly, the 96-well microtiter plate (Nunc, Naperville, IL, USA) was coated with 100 $\mu$ L/well (10 $\mu$ g/mL) of the FimH antigen in 0.05 M carbonate buffer (pH 9.6) and incubated overnight at 4 ${}^{\circ}$ C. The wells washed three times with PBS containing 0.05% Tween 20 (washing buffer) and blocked for 1 h at 37 ${}^{\circ}$ C with 0.05% BSA in PBS (blocking buffer). The plates were washed with washing buffer, and 100 $\mu$ l of 1/50 to 1/6400 diluted sera were added to individual wells and incubated at 37 ${}^{\circ}$ C for 2 h. The wells were washed five times with washing buffer, and incubated for 2 h with 100 $\mu$ l of 1/10000 dilution of HRP-conjugated anti mouse (Sigma, USA). After washing, 3, 3’, 5, 5’-tetramethylbenzidine (TMB)-ELISA solution was used as a substrate. Finally, the reaction was stopped with adding 100 $\mu$ l of 2 M H ${}_{2}$ SO ${}_{4}$ , and the OD was measured at 450 nm by a microplate spectrophotometer. In addition, specific IgG1and IgG2a subclasses were assessed at dilution of 1/50 of experimental sera with goat anti-mouse IgG1and IgG2a secondary antibodies (Sigma, USA) according to the manufacture’s protocol.

2.8 Determination of cytokine concentrations

Two weeks after the final immunization, a total number of 3 $\times$ 10 ${}^{6}$ splenocytes were seeded into each well of a 24-well plate and stimulated with 10 $\mu$ g/ml of FimH protein and incubated at 37 ${}^{\circ}$ C in 5% CO ${}_{2}$ . Three days later, supernatants were collected and interferon- $\gamma$ (IFN- $\gamma$ ) and interleukine-4 (IL-4) cytokines were assessed through commercial ELISA Kits (Mabtech, Sweden) according to the manufacturer’s instructions. The quantity of cytokines was expressed as pg/ml according to the related standard curve. Every cytokine was tested two times.

2.9 Mouse model of ascending UTI

Mouse models of UTI were used to evaluate the protective ability of FimH immunization [18, 19]. For the mouse UTI model, BALB/c mice were intraperitoneally anesthetized with a cocktail of ketamine (100 mg/kg), xylazine (10 mg/kg), and acepromazine (2.5 mg/kg) and the bladder was emptied by gentle pressure on the abdomen. The median infection dose for transurethral inoculation of E. coli was determined previously as 10 ${}^{8}$ CFU/mouse (35). Uropathogenic E. coli containing FimH gene was cultured on LB agar slants for 18 h at 37 ${}^{\circ}$ C; the resulting bacterial lawns were suspended in PBS. To ensure infection, mice were inoculated with 50 $\mu$ l of PBS containing 10 ${}^{8}$ CFU of E. coli via transurethral catheterization using a sterile polyethylene catheter (Intramedic, Becton Dickinson, MD, USA). One, two and three days after challenge, surviving mice were sacrificed and bacteria in the bladder and kidneys were quantitatively cultured on LB agar to measure the number of CFU per gram of tissue. The lower limit of detection in this assay was 10 ${}^{2}$ CFU/g of tissue. Thus, this value (10 ${}^{2}$ CFU/g of tissue) was assigned to samples with an undetectable level of colonization.

2.10 Statistical analysis

All experiments were performed in triplicate, and the data was expressed as means $\pm$ S.D. for each experiment. All statistical analyses were carried out by ANOVA followed by Tukey’s test and Microsoft Excel 2016 software. In all of the cases, p values $<$ 0.05 were considered to be statistically significant.

3. Results and discussion

3.1 Construction of fimH gene

The fimH gene was amplified by PCR using E. coli 35218 chromosomal DNA as the template. The PCR conditions were optimized for amplification of fimH gene. Electrophoresis of PCR products showed that the length of PCR fragment of fimH genes was approximately 905 bp (Fig. 1). FimH gene has some nucleotides variation among enterobacteriaceae like E. coli. Protein sequences of E. coli UTI89 strain (native fimH, reference gene) and fimH synthesized from E. coli 35218 strain aligned using ClustalW (http://www. ebi.ac.uk/Tools/clustalw2). The sequence of E. coli 35218 fimH gene was shown more than 97% identity to other fimH sequence reports in GenBank [21]. Seven point mutations in 35218 fimH gene and one changed amino acid in 35218 fimH protein sequence (Table 1) was found.

Table 1
Result of FimH protein alignment

4913555-49144 fimH35218	1ATGAAACGAGTTATTACCCTGTTTGCTGTACTGCTGATGGGCTGGTCGGTAAATGCC
	1ATGAAACGAGTTATTACCCTGTTTGCTGTACTGCTGATGGGCTGGTCGGTAAATGCC
4913555-49144 fimH35218	58TGGTCATTCGCCTGTAAAACCGCCAATGGTACCGCAATCCCTATTGGCGGTGGCAGC
	58TGGTCATTCGCCTGTAAAACCGCCAATGGTACCGCTATCCCTATTGGCGGTGGCAGC
4913555-49144 fimH35218	115GCCAATGTTTATGTAAACCTTGCGCCTGCCGTGAATGTGGGGCAAAACCTGGTCGTA
	115GCCAATGTTTATGTAAACCTTGCGCCTGCCGTGAATGTGGGGCAAAACCTGGTCGTG
4913555-49144 fimH35218	172GATCTTTCGACGCAAATCTTTTGCCATAACGATTACCCAGAAACCATTACAGACTAT
	172GATCTTTCGACGCAAATCTTTTGCCATAACGATTACCCGGAAACCATTACAGACTAT
4913555-49144 fimH35218	229GTCACACTGCAACGAGGTGCGGCTTATGGCGGCGTGTTATCTAGTTTTTCCGGGACC
	229GTCACACTGCAACGAGGTTCGGCTTATGGCGGCGTGTTATCTAGTTTTTCCGGGACC
4913555-49144 fimH35218	286GTAAAATATAATGGCAGTAGCTATCCTTTCCCTACTACCAGCGAAACGCCGCGGGTT
	286GTAAAATATAATGGCAGTAGCTATCCTTTCCCTACTACCAGCGAAACGCCGCGGGTT
4913555-49144 fimH35218	343GTTTATAATTCGAGAACGGATAAGCCGTGGCCGGTGGCGCTTTATTTGACGCCGGTG
	343GTTTATAATTCGAGAACGGATAAGCCGTGGCCGGTGGCGCTTTATTTGACGCCTGTG
4913555-49144 fimH35218	400AGCAGTGCGGGGGGAGTGGCGATTAAAGCTGGCTCATTAATTGCCGTGCTTATTTTG
	400AGCAGTGCGGGGGGAGTGGCGATTAAAGCTGGCTCATTAATTGCCGTGCTTATTTTG
4913555-49144 fimH35218	457CGACAGACCAACAACTATAACAGCGATGATTTCCAGTTTGTGTGGAATATTTACGCC
	457CGACAGACCAACAACTATAACAGCGATGATTTCCAGTTTGTGTGGAATATTTACGCC
4913555-49144 fimH35218	514AATAATGATGTGGTGGTGCCCACTGGCGGCTGCGATGTTTCTGCTCGTGATGTCACC
	514AATAATGATGTGGTGGTGCCCACTGGCGGCTGTGATGTTTCTGCTCGTGATGTCACC
4913555-49144 fimH35218	571GTTACTCTGCCGGACTACCCTGGTTCAGTGCCGATTCCTCTTACCGTTTATTGTGCGA
	571GTTACTTTGCCGGACTACCCTGGTTCAGTGCCGATTCCTCTTACCGTTTATTGTGCGA
4913555-49144 fimH35218	628AAAGCCAAAACCTGGGGTATTACCTCTCCGGCACAACCGCAGATGCGGGCAACTCG
	628AAAGCCAAAACCTGGGGTATTACCTCTCCGGCACAACCGCAGATGCGGGCAACTCG
4913555-49144 fimH35218	685ATTTTCACCAATACCGCGTCGTTTTCACCCGCGCAGGGCGTCGGCGTACAGTTGACG
	685ATTTTCACCAATACCGCGTCGTTTTCACCCGCGCAGGGCGTCGGCGTACAGTTGACG
4913555-49144 fimH35218	742CGCAACGGTACGATTATTCCAGCGAATAACACGGTATCGTTAGGAGCAGTAGGGAC
	742CGCAACGGTACGATTATTCCAGCGAATAACACGGTATCGTTAGGAGCAGTAGGGAC
4913555-49144 fimH35218	799TTCGGCGGTAAGTCTGGGATTAACGGCAAATTACGCACGTACCGGAGGGCAGGTGA
	799TTCGGCGGTAAGTCTGGGATTAACGGCAAATTACGCACGTACCGGAGGGCAGGTGA
4913555-49144 fimH35218	856CTGCAGGGAATGTGCAATCGATTATTGGCGTGACTTTTGTTTATCAATAA
	856CTGCAGGGAATGTGCAATCGATTATTGGCGTGACTTTTGTTTATCAATAA

Figure 1.

PCR amplification of fimH gene. Lane 1–4: products of fimH gene (905 bp); Mw: Molecular weight marker (1 kb ladder DNA).

Figure 2.

Lane 1: pBluescriptIISK cloning vector without fimH gene. Lane 2, 3: pBluescriptIISK cloning vector with fimH gene.

The amplified gene was blunt end fragment, purified from gel and inserted into EcoRV digested pBluescriptIISK cloning vector yielding recombinant plasmids, designated as pBlue/fimH gene plasmid. The plasmid were transformed to E. coli TOP10F’ strain by chemical methods (CaCl ${}_{2}$ ), extracted and purified using a plasmid Miniprep Kit (GeneJET, Fermentas, Germany). The qualities of resulting plasmids were assessed by 0.8% agarose gel electrophoresis (Fig. 2). Figure 2 confirmed the insertion of FimH gene into the plasmid.

Figure 3.

Result of PCR on pET23a/fimH as template.

Figure 4.

CATATG: NdeI restriction site (ATG used as start codon), FimH lectin domain sequence, CACCACCACCACCACCAC: His-tag sequence, TAATGA: Two stop codons, GAATTC: EcoRI restriction site, Total sequence: 504 bp.

3.2 Construction of the fimH

{}_{1-156}

(fimH lectin domain) expression plasmid

After confirmation of pBluescript/fimH via electrophoresis and sequencing, the region encoding the N-terminal half of the FimH adhesion (i.e. the signal peptide and residues 1–156 of the mature protein) was amplified by PCR using two specific primers (Fig. 3). The 504 bp PCR product (Fig. 4) inserted to pET23a expression vector. The primer contained an NdeI restriction site, fimH lectin domain sequence, His-tag sequence, two stop codons, and EcoRI restriction site. This resulted in a construct consisting of the first half of FimH (i.e. the receptor-recognition domain) fused to a histidine tag. We refer to the protein encoded by this truncated fimH gene as FimH ${}_{\text{156-His}}$ . The amplified PCR product ligated into pET23a.

The resultant plasmid (pET23a/fimH) confirmed through sequencing. The result of sequencing of inserted fimH lectin domain gene to pET23a showed right frame of it with kozak sequence.

3.3 Expression and purification of FimH ${}_{1-156}$

The FimH ${}_{\text{156-His}}$ protein was expressed in E. coli origami (DE3) by induction with IPTG. The level of expression of FimH were analyzed by SDS-PAGE then by western blotting using anti-His antibody. The results of SDS-PAGE and western blot of the FimH, are shown in Fig. 5 and 6. The analysis of SDS-PAGE and western blot of the proteins showed single bands at the size of approximately 17 kDa for Fim ${}_{\text{H156-His}}$ protein.

Figure 5.

Analysis of expressed products of FimH by 15% SDS-PAGE. Lane 1: Uninduced construct; Lane 2–9: pET-FimH induced by IPTG after 0.5, 1, 2, 3, 4, 5, 6, 7 h; Lane 10:17 kDa pure protein; Mw: Protein marker.

Figure 6.

Western blot analysis of FimH imduced by IPTG after 1 h; Lane 2: Uninduced construct.

Periplasmic fractions were prepared from a 2 L culture and the Fim ${}_{\text{H156-His}}$ protein was purified using a Ni-NTA affinity matrix (Qiagen). A single protein with an expected size of approximately 17 kDa was obtained after purification on the Ni-NTA affinity matrix. Interestingly, we obtained higher yields of FimH ${}_{\text{156-His}}$ in the E. coli origami (DE3) comparable to that of well-documented strain BL21. Previous reports have demonstrated that expression of the fimH gene leads to the formation of naturally occurring FimH truncates with inherent K-D-mannose recognition [21, 22]. Here, no degradation of the FimH ${}_{\text{156-His}}$ protein was observed, suggesting that processing sites are not contained within the FimH ${}_{\text{156-His}}$ protein.

3.4 Endotoxin level in the purified fimH protein

To assess whether lipopolysaccharide (LPS) levels in the range of 0.1–1 EU are actually present in FimH recombinant protein, LAL assay was performed. The results showed that the endotoxin level was less than 0.1 EU/ml. It can be hypothesized that the contaminating endotoxins were primarily associated, and thus removed, with the histidine tags [23, 24]. For this reason, we devised a strategy in which we separated the purified protein from the histidine tags and the contaminating endotoxins.

3.5 Lymphocyte proliferation

The results of lymphocyte proliferation indicated that the candidate vaccine, FimH protein, adjuvanted with alum and freund’s resulted in an increase in lymphocyte proliferation as compared to control group ( $p<$ 0.005, $p=$ 0.0408 respectively). In addition, the FimH/alum immunization of mice led to more increment of lymphocyte proliferation in comparison to the FimH/freund’s immunized group and this increase was significant ( $p=$ 0.0011) (Fig. 7). It can be suggested that this candidate primarily induces Th1 type immune responses in the test mice.

Figure 7.

Proliferation of lymphocytes. Stimulation index (SI) was calculated based on cell proliferation, as determined using the ELISA-BrdU assay. Data are presented as the mean $\pm$ standard error of the mean. Statistical significance of differences is illustrated as follows: ** $P<$ 0.01.

3.6 Humoral immune responses and isotyping

Humoral immune response was determined with indirect ELISA method. As depicted in Fig. 8, significantly increased levels of total IgG at different dilutions were present in FimH/Freund and FimH/alum immunized mice compared to the control group ( $P<$ 0.05). But no significant differences of IgG were observed between the immunized FimH/freund and FimH/ Alum groups ( $P=$ 0.011).

Figure 8.

Specific humoral immune response monitoring against FimH after immunization periods. Sera of individual mice were collected and specific IgG titer was evaluated with ELISA.

Furthermore, we characterized the specific isotypes of antibodies that were induced. Monitoring of IgG titer during the study revealed significant difference between FimH/Alum, FimH/freund’s and PBS groups ( $P<$ 0.05) (Fig. 9). However, No significant differences were observed in the IgG2a level between FimH/alum and FimH/freund’s immunized groups ( $p$ $=$ 0.96).

Figure 9.

The IgG2a isotype responses indicated that immunization with FimH/alum as well as FimH/freund’s increased IgG1 and IgG2a when compared to the control group ( $p=$ 0.025).

Figure 10.

IFN- $\gamma$ and IL-4 cytokine assay of immunized mouse groups.

Various studies showed the role of humoral immune responses in the resistance to the infection [26, 25]. In fact, antibodies masking the adhesion molecules on UPEC are able to inhibit the attachment to the host cells and abrogate colonization and subsequently the infection [27] and FimH, as an adhesion molecule, can induce strong antibody responses with increased IgG1 and IgG2a classes. Taken together, in the preset study we have shown the potency of the FimH molecule as an immunogen in the induction of humoral and cellular immune responses. However, in the near future, the FimH vaccine using the surface display strategy would be evaluated in the experimental UTI challenges.

3.7 Cytokine assay

Assessment of the IFN- $\gamma$ cytokine in the experimental groups showed that immunization with FimH increased the IFN- $\gamma$ cytokine as compared to the control PBS group ( $P=$ 0.0008) (Fig. 10). However, No significant difference was observed between alum and freund’s adjuvanted groups in the induction of the IL-4 cytokine (Fig. 10). IL-4 has been shown to be crucial for control of infections and plays a crucial role in preventing the development of strong IFN- $\gamma$ -driven responses [28]. However, there was no significantly higher concentrations of IL-4 observed in groups immunized with FimH and control group. IFN- $\gamma$ is reported to be associated with protective immune responses [29].

Figure 11.

Effect of vaccination on pathogenic E. coli ability to cause urinary tract infection in mice.

3.8 Protection studies in mouse model

In the UTI mouse model, immunized and naive mice were intraurethrally challenged with 10 ${}^{8}$ CFU of uropathogenic E. coli to determine the impact of vaccination with FimH protein on bacterial loads in urinary system organs (bladder and kidney) (Fig. 11). Seven days after challenge, bacteria in bladder and kidneys of surviving mice were quantitatively cultured. Significant decreases in colonization were achieved by (bladder, P 0.007; kidney, P 0.03) immunization. Our data indicated that FimH vaccine were effective in protecting mice. The protection of mice by the FimH vaccine from E. coli UTI is comparable to the protection by the truncate MrpH vaccine from P. mirabilis UTI [30]. For comparison to this study, if one uses a mean bladder weight of 0.5 g to convert the data of Langermann et al. [31], then protection of the bladder from colonization following challenge of vaccinated animals was nearly identical for E. coli and P. mirabilis. For the bladder, the FimH E. coli vaccine appeared to offer better protection. This is especially important since uropathogenic E. coli localizes to the bladder and can cause serious histological damage to these organs, even after a single infection. The protection from E. coli UTI was achieved by systemic immunization with the FimH vaccine which inducing a strong serum IgG response.

4. Conclusions

Urinary tract infection (UTI) is one of the most common infections diagnosed in patients. Uropathogenic Escherichia coli (UPEC) are the most common patho-gen found in urinary tract infection, being isolated in around 80% of UTI cases [32]. The emergence of antibiotic resistance in UPEC strains in the world is the major cause for an increasing requirement for vaccine development against UTI [8].

One of the important criteria for an ideal vaccine target against UPEC is its wide distribution among clinical UPEC isolates. Some studies showed that FimH is highly conserved among UPEC strains [33]. We selected the pET23a expression system for expression of FimH, protein. The T7 promoter of the expression system results in robust expression of the target gene. The results of immune response assay confirmed the ability of FimH to induction of Th1 cytokine profile, and the possible potency of this vaccine to raising the protective immune response against UTI. In mouse models, immunization with FimH truncates have been shown to prevent urogenital mucosal infection by E. coli. In addition, passive systemic administration of immune sera directed against FimH also resulted in reduced colonization of the urinary bladder.

Finally, it can be concluded that our FimH histidine-tagged protein could potentially be exploited in the development of a vaccine to prevent recurrent and acute infections of the urogenital mucosa.

Footnotes

Acknowledgments

The authors would like to thank the research council of Islamic Azad University for the financial support of this investigation.

Conflict of interest

The authors do not declare any conflict of interest.

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Construction and development of FimH lectin domain for rising immune response after injection by uropathogenic E. coli

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Amplification of E. colifimH gene

2.2 Expression of the fimH genes

2.3 SDS-PAGE and western blot

2.4 Purification of the fimH-his protein

2.5 Mice and immunization experiments

2.6 Lymphocyte proliferation assay

2.7 Total antibody ELISA and determination of igG1, igG2a subclasses

2.8 Determination of cytokine concentrations

2.9 Mouse model of ascending UTI

2.10 Statistical analysis

3. Results and discussion

3.1 Construction of fimH gene

Table 1 Result of FimH protein alignment

3.3 Expression and purification of FimH 1 - 156

3.5 Lymphocyte proliferation

4. Conclusions

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Result of FimH protein alignment

3.3 Expression and purification of FimH ${}_{1-156}$