Studying the interaction between uropathogenic Escherichia coli (UPEC) and uroepithelial cells is important in elucidating the pathogenesis of urinary tract infection. In this study, the African green monkey kidney cells (Vero), human kidney carcinoma cells (Ketr-3) and bladder carcinoma cells (EJ) were infected by UPEC132, a clinical strain isolated from Tianjin, China, and were compared for their capacities to allow the adherence and invasion by this strain. The results revealed that all these cell lines could be attached and invaded by UPEC132. The adherence rates for Vero, Ketr-3 and EJ cells were (49.20 ± 7.55)%, (55.22 ± 4.09)% and (73.20 ± 5.26)%, respectively, and invasion frequencies were (2.61 ± 0.32)×10-3, (3.00 ± 0.34)×10-3 and (3.25 ± 0.20)×10-3, respectively. The statistical analysis showed that the adherence rate for EJ cells was significantly higher than those for the other two cell lines (P<0.05), and the invasion frequencies for EJ and Ketr-3 cells had no statistical differences (P>0.05) but were higher than that for Vero cells (P<0.05). Three cell lines were detected for the receptors for P pili of UPEC by using indirect immunofluorescence. The results showed that receptors existed on the surfaces of all cell lines, and the highest distribution was found on the surface of EJ cells. Additionally, the invasion of EJ cells by recombinant UPEC132/pSELECT-GFP could be directly visualized using confocal microscopy. These data strongly implicated that EJ cells could be more easily infected by UPEC132 than the other cells, and thus could serve as a good experimental target for further investigation of UPEC infection.
GE Xin DONG Jie CHEN JinYing YAO Ping GU Chao YANG DongJing
Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM) with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.
Uropathogenic Escherichia coli(UPEC) is the most common causative organism of human urinary tract infection(UTI).Several UPEC virulence factors have been identified,but more are yet to be found.We previously identified a novel 789-bp-long DNA fragment(named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique.In the present study,we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity.Through bioinformatic analysis,we predicted that this gene is a 1311-bp open reading frame(ORF),which we designated ORFR049(GenBank accession No.:EF488001).We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography.Using mouse immunosera generated by the purified protein,we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting.To test the potential of this protein as a vaccine candidate,we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract.The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group.However,the degree of renal pathological damage was not significantly improved in the immunized mice.Our study has identified a novel gene of UPEC which can generate protective immunity against UTI.This novel gene provides a promising new vaccine candidate.
CHEN JinYingGE XinZHANG YuMeiLIN XuZHANG WeiYANG Xi
