Production And Characterization Of Enteropathogenic Escherichia Coli Ghosts

Enteropathogenic Escherichia coli (EHEC) is an important food borne pathogen capable of causing bloody. EPEC infection was mainly transmitted by fecal–oral, with contaminated hands, foods or water. In developed countries, EPEC once caused frequent outbreaks of infant diarrhea. The EPEC infection outbreaks were often explosive, with up to 50% mortality. In developing countries, about 30~40 % of infants diarrhea caused by EPEC, especially in the 0-6 month-old infants. At present, for EPEC infection mainly treated with antibiotics. However, antibiotic treatment may cause some side-effects. The best method to prevent EPEC infection is to develop a vaccine.Bacterial ghost is a new type of bacterial vaccines developed in recent years. Bacterial ghosts usually produced by PhiX174 phage protein E at the bacterial surface to form a pore-like structure, and bacterial cell contents exhausted through the pore-like structure to form the non-cytoplasmic non-active bacteria.Bacteria ghosts were produced by non-denatured methods. The natural surface antigen and cell morphology of bacteria were well-preserved. Bacteria ghosts also have adjuvant activity on its own. Furthermore, bacteria ghosts could be used as vaccine vector. Bacteria ghosts production process is simple and safe.PhiX174 phage lysis gene E encodes a 91 amino acid membrane protein. Protein E can integrate internal and external membrane of Gram-negative bacteria, resulting in a hole-like channel formed on the surface of bacteria. The diameter of hole-like structure of bacteria ghosts is between the range of 40-200nm. Cytoplasmic of bacterial was exhausted through this pore-like structure to form the non-cytoplasmic non-active bacteria. But the membrane structure of bacteria was well-preserved.Staphylococcal aureus nuclease A (SNA), also known as Micrococcal nuclease enzyme, is a non-specific nuclease secreted by Staphylococcus aureus. SNA can degrade the host bacterial DNA. SNA has a strong ability to degrade single-stranded or double-stranded DNA or RNA. It can degrade bacteria DNA into small fragments less than 100bp.Plasmid pBV220 has a thermo-sensitive transcriptional control systemλpL/pR-cI857, which can inhibit protein expression at 28℃. The protein expression could be induced by transfer the temperature from 28 to 39~42℃.To develop a vaccine against Enteropathogenic E. coli, in this study, we intend to use the temperature-sensitive plasmid pBV220 to express PhiX174 lytic gene E and staphylococcal nuclease A to produce EPEC ghosts. And we further study the biological and immunological characteristics of the EPEC ghosts.A pair of primers was designed according to PhiX174 phage E gene sequence published in GenBank. The lysis gene E was amplified by PCR from PhiX174 phage DNA by use of primers LE-F and LE-R. PCR product was cloned into vector pMD18-T and then subcloned into pBV220 downstream of theλPL/PR-cI857 regulatory system between sites EcoRI and BamHI to form the temperature-sensitive bacteriolysis plasmid pBV220::E. The recombinant plasmid pBV220::E was transformed into host cell DH5α. The lysis curve and lysis efficiency of plasmid pBV220::E to host cell DH5αwere studied. Results showed recombinant plasmid pBV220::E could lysis DH5αafter the temperature shift from 28℃to 42℃. The lysis rate of culture was 99.76%.In order to improve the efficiency of generation of bacteria ghosts, Staphylococcal nuclease A (SNA) was amplified from Staphyloccocus aureus by PCR. Temperature regulation fragment CI-P was amplified from plasmid pBV220. These two fragments were ligated to form fusion fragment CI-P-SNA by PCR. The fusion fragment CI-P-SNA was subcloned into downstream sites BamHI and PstI of lysis gene E to generate a recombinant plasmid pBV220::E::CI-P-SNA. This recombinant plasmid was transformed into host cell DH5α. The lysis curve and lysis efficiency of plasmid pBV220::E::CI-P-SNA to host cell DH5αwere studied. Results showed recombinant plasmid pBV220::E::CI-P-SNA could lysis DH5αafter the temperature shift from 28℃to 42℃. The lysis rate of culture was 99.53%.Recombinant plasmid pBV220::E and pBV220::E::CI-P-SNA were transformed into EPEC reference strain E2348/69 (O127:H6). The resultant recombinant strains were named as F158 and F159, respectively.The lysis curve, lysis efficiency and bacterial ghosts morphology of strains F158 and F159 were studied. The results showed that strains F158 and F159 were grown at 28℃until mid log-phase, followed by incubation at 42℃to induce the expression of gene E and SNA to generate EPEC bacterial ghosts. The OD600 of culture began to decline 20 min after the temperature shift from 28℃to 42℃. The lysis rate of F158 and F159 culture were 99.92% and 99.95%, respectively. Electron microscopy observations indicated that EPEC ghosts have basic cell morphology of E. coli. The EPEC ghosts were shown to be intact cells with contents released to extracellular region. However, as bacterial cytoplasmic flow outside of bacteria, EPEC ghosts surface significantly shrink. There are few living bacteria surviving in EPEC ghosts. The living bacteria surviving in EPEC ghosts could be completely killed after treatment by freezing and thawing in 5% or 10% NaCl solution.This study has also established an animal model of mice infected with EPEC. We studied the safety and immune effects of EPEC ghosts using mice infection model. EPEC ghosts produced from F158 and F159 by temperature induction were used to infect mice. Negative control mice,inoculated with F158 or F159 EPEC ghosts grew normally. Mice inoculated with wild-type strain EPEC E2348/69 showed rapid weight loss and all mice were died within 4 days. The results showed that EPEC ghosts are safe to mice.Experiments of immunity and challenge showed that all mice of non-immunized control group died (20/20), 13 mice immunized with F158 EPEC ghosts were survive 13/20, 16 mice immunized with F159 EPEC ghosts were survive16/20. It is suggestion that EPEC ghosts have good immune protective effect.We can conclude from above study that recombinant plasmid pBV220::E and pBV220::E::CI-P-SNA could be used to generate EPEC ghosts, EPEC ghosts is safety and have good immune protection. This study showed that EPEC ghosts could be used as candidate vaccine against EPEC infection.