Construction Of MLT-producing Mutant Strains From Enterotoxigenic Escherichia Coli And Their Main Biological Properties

Enterotoxigenic Escherichia coli?ETEC?is a leading cause of diarrhea in travelers,children,and juvenile animals,which causes neonatal and post-weaning diarrhea in piglets,creating a significant threat to swine industry worldwide and resulting in huge economic losses.The emergence of drug-resistant strains has significantly reduced the efficacy of antibiotics.Therefore,new and efficient drugs or vaccines are urgently required to protect the suckling piglets.Oral vaccine has the advantages of inducing intestinal local immunity,humoral immunity and cellular immunity,and provides new ideas for the developments of piglet colibacillosis vaccines.Enterotoxins produced by ETEC mainly include heat-labile enterotoxin?LT?and heat-stable enterotoxin?ST?,which are the main pathogenic factors.Although LT has good antigenicity,its toxicity limits its application on animal vaccine.Studies on attenuated mutant LT?mLT?have been reported,but the construction and biological properties of different mLT mutant strains are still lack of systematic researches.In this study,three mLT-producing E.coli mutant strains were constructed,aiming to screen ideal candidates for the oral vaccines against E.coli diarrhea in piglets by systematically comparing their main biological properties.In this study,E.coli C83903?K88,STb,LT?was used as the initial strain,and its est B gene was deleted by using the CRISPR/Cas9 dual plasmid gene editing system and the E.coli C83903?35?est B was obtained.Then some site-directed mutations of the gene elt encoding LT were operated and the E.coli mLT-S63K,E.coli mLT-A72R and E.coli mLT-R192G were obtained.The experimentally test results showed that the three mutant strains were genetically stable after 50 consecutive passages;the biological properties such as bacterial growth curve,fimbrial growth capacity,adhesion ability to IPEC-J2 cells and antibiotic sensitivity showed no significant difference with the initial strain,and the mutant strains were well-tolerated in gastric juice environment with p H>2.5 and in 0.3%bile environment.In this study,E.coli p ET-32a-S1-BL21 and E.coli p ET-32a-S2-BL21 recombinant strains were constructed,and the recombinant proteins LTA-S1 and LTA-S2 were expressed and purified.The rabbit anti-LTA-S1 and rabbit anti-LTA-S2 serum were prepared by immunizing rabbits.The rabbit anti-LTA-S1 serum was used for the subsequent detection of LT and mLT because of its higher titer.Results of pathological sections and ligation of rabbit ileum showed that three different mLTs were enterotoxic.Among them,E.coli mLT-S63K produced the weakest enterotoxicity of mLT and caused minimal damage to intestinal tissue.The results of Y1 cytotoxicity test showed that the enterotoxicity of mLT-S63K on Y1 cells was also the weakest.The virulence titer is 10^3.48TCID₅₀/0.1m L,which was 1/10 000 of the LT toxicity produced by the E.coli C83903(10^7.61TCID₅₀/0.1m L).Five-weeks-old BALB/c mice were randomly divided into 6 groups?A,B,C,D,E,F?,25 in each group,and were fed with 0.3 m L sterile PBS and 3×10¹⁰CFU of E.coli C83903?35?est B?35?elt?,E.coli C83903?35?est B,E.coli mLT-S63K,E.coli mLT-A72R and E.coli mLT-R192G for 3 consecutive days by gavage,3 immunizations at two-week intervals.Compared with the control group,the test results showed that the mutant strains had no effect on the daily appetite and body weight of the mice,no damage to the ileal tissues and all of them had good oral safety;The levels of specific Ig G antibody in serum and specific s Ig A antibody in intestine mucus were detected by indirect-ELISA.The results showed that on the 14th day after the first immunization,specific Ig G and s Ig A against the LTA-S1 protein were detected from the serum and intestinal mucus of the experimental group,and the antibody levels were significantly higher than the control group?p<0.05?.The Ig G level detected in group E.coli mLT-R192G on the 35th day was significantly higher than other groups?p<0.05?.In group E.coli mLT-S63K,the s Ig A level detected on the 42nd day was significantly higher than that of the other groups?p<0.05?,and it could maintain a high level within 2 weeks after the third immunization.The test results of specific Ig G and s Ig A antibody levels against K88 pili and E.coli C83903 strain showed that specific s Ig A could be detected in the intestinal mucus of some immunized groups on the 7th day after the first immunization,of which group E.coli mLT-A72R at the fastest rate.The s Ig A antibody level of group E.coli mLT-A72R was significantly higher than the other groups on the 21st day and could maintain a high level;The K88-specific Ig G in group E.coli mLT-S63K has been at a relatively high level since the 21st day,and it was still significantly higher than the other groups at 42nd day?p<0.05?.For the specific antibody against E.coli C83903 strain,the Ig G level of the group E.coli mLT-S63K on the 35th day was significantly higher than that of the other experimental groups?p<0.05?.The levels of s Ig A in the intestinal mucus of the group E.coli mLT-S63K and the E.coli mLT-R192G were significantly higher than that of the other experimental groups?p<0.05?,and could maintain a high level on the 42nd day.The results of immunohistochemistry experiments showed that on the 35th day after the first immunization,the Peyer's lymph node tissue sections of mice in the group E.coli mLT-S63K and the group E.coli mLT-R192G showed significantly more Ig A positive cells in number.The results of splenic lymphocyte proliferation experiment and flow cytometry showed that the number of IFN-?and IL-4 positive splenic lymphocytes was significantly higher than that of the control group,and the ratio of IFN-?/IL-4 was less than 1.The determination results of IFN-?,IL-4 and IL-17 serum cytokines levels showed that oral mutant strains could effectively stimulate the body to produce Th1,Th2 and Th17 type cellular immunity,and the expression level of IL-4 in serum was higher than the others.The detection results of lymphocyte subsets indicated that the mutant strain induced mainly Th2 type cell immunity.The results of antibody neutralizing activity test results showed that the Ig G and s Ig A of the immunized group had neutralizing activity of LT,the serum neutralizing antibody titers of the three groups of mutants were 1:89.1,and the neutralizing antibody titers of intestinal mucus were 1:44.7,both higher than 1:53.7 and 1:26.9 in group E.coli C83903?35?est B,respectively.In summary,the three mLT mutants constructed in this study are genetically stable,and their main biological properties are not significantly effected after gene editing operations.The biological properties of the mutant strains are suitable for the basic conditions of oral vaccine candidate strains.The oral immunization of the mutant strains can induce local mucosal immunity,humoral immunity and cellular immunity.The E.coli mLT-S63K strain has the minimal toxicity and the best immunogenicity,which can be used as a candidate strain for the oral vaccine in subsequent research.This study provides experimental data and candidate strains for a new vaccine against E.coli diarrhea in piglets.