The Study Of Anti-infection Effect Of Human Beta-defensin-2 Gene In Urinary Tract Infections

Objective:Human beta-defensin is a family of cationic antimicrobial peptides that are widely expressed at epithelia and mucosal surface of human body. These small cationic antimicrobial peptides exhibit broad-spectrum activity against bacteria, fungi and some enveloped virus, which play important roles in the defense against microbial invasion. The purpose of this study was to evaluate the potentiality of urothelial cells to be transfected by eukaryotic expression vector of hBD-2 to produce hBD-2 which possess antimicrobial activity in vitro and in vivo. At the same time, using a UTI rat model to investigate the role of transgene hBD-2 to prevent and control bacterial infection in the urinary tract. The main objective of the present study was to assess the potentiality and efficacy of hBD2 gene transfection as a therapeutic tool for future antimicrobial gene therapy of UTI.Methods:The total RNA was isolated from human cervical carcinoma tissue and hBD-2 cDNA fragments were obtained by RT-PCR amplification with specific primers. HBD-2 cDNA containing the entire coding region previously cloned into the pGEM-T-Easy vector was released with EcoR I restriction enzymes and inserted into a eukaryotic expressing vector pCAGG. The eukaryotic expressing vector carrying the hBD-2 gene was designated pCAGG-hBD-2 and the inserted hBD-2 sequence confirmed with a standard DNA sequencing method. Transient transfection was performed using a cationic Liposome-TransFastTM Transfection Reagent. The level of secreted hBD2 in cell supernatants and rats'urine was checked by ELISA. The expression of the transgene hBD-2 in situ was confirmed by immunohistochemistry. Expression of the transgene hBD-2 in transfected cells and rats'bladders were also confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. The in vitro antimicrobial activity of secreted hBD-2 from pCAGG-hBD2 transfected T24 supernatants.was measured by colony-forming unit (cfu) assay. We then used a UTI rat model to test hBD-2 antimicrobial activities in vivo. Either recombinant pCAGG-hBD2 or PBS was administered intravesically to rats 48 h before establishing UTI. Then the median bacterial counts in the bladders, urine and kidneys of rats both from the pCAGG-hBD2 or PBS administered were measured. The inflammatory responses in the urinary tract during infection were also investigated.Results:1. DNA sequencing analysis demonstrated that the hBD-2 was correctly inserted into the eukaryotic expression vector pCAGG, and the successfully constructed eukaryotic expression vector pCAGG-hBD-2 could be used to do gene transfection.2. RT-PCR revealed that amplified hBD-2 cDNA fragments were present in the samples derived from T24 cells and bladders bearing pCAGG-hBD-2 but not in those from carrying control vector cells and bladders. The expression of hBD2 protein in transduced T24 cells and bladders was also confirmed by Western blotting analysis. Only Cells and bladders bearing pCAGG-hBD-2 expressed transgene hBD-2. 3. ELISA assay results showed that concentrations of hBD2 were 36.5±3.2 ng/106 cells and 4.77±1.4 ng/mL respectively in the supernatants of T24 and rat's urine after pCAGG-hBD2 transduction, whereas their control vector transfected counterparts did not secrete any detectable hBD-2.4. Immunohistochemistry revealed that in situ gene transfer to bladder could be accomplished via intravesical instillation of plasmid DNA/liposome after a single 2 h transfection. The hBD-2 expression can be detected in the entire epithelial layer of the bladders transfected with hBD2.5. Cell culture supernatants from hBD-2 transfected T24 cells exhibited a rapid and prolonged bactericidal activity against clinical isolates of UPEC strain UTI89 and K. pneumoniae TOP52.6. Either recombinant pCAGG-hBD2 or PBS was administered intravesically to rats 48 h before establishing UTI. Numbers of bacterial colony-forming unit (CFU) in urine, bladders and kidneys from hBD2 transfected UTI rats were significantly lower than those from the control UTI animals at 24,36, and 72 h after infection (P<0.05). In addition, in vivo expression of hBD-2 could reduce mucosa damage, interstitium edema and inflammatory cells infiltration in UTI animals.Conclusions:This study demonstrated that the recombinant expression vector of hBD-2 could be effectively transferred into bladder epithelial cells by a liposome-mediated plasmid DNA transfection protocol with a significant level of transgene expression of hBD-2 and well tolerated. The transgene hBD2 possessed obvious antimicrobial activity in vitro and vivo. Moreover, overexpression of hBD-2 modulated inflammation during the early course of bladder injury and improved the outcome of UTI. Intravesical introduction of exogenous antimicrobial peptide genes is a potentially useful therapeutic modality for the UTI or rUTI.