Applied Microbiology and Biotechnology

Generation of an attenuated-strain oral vaccine candidate using a novel double

selection platform in Escherichia coli

Wenxin Liu1, Chaowen Yuan

1, Jun Bao

2, 3, Weikun Guan

1, Zhiteng Zhao

1, Xingyue

Li1, Jie Tang

1, Dandan Li

4, Dongfang Shi

1, 3*

1

Department of Preventive Veterinary Medicine, College of Veterinary Medicine,

Northeast Agricultural University, Harbin, Heilongjiang 150030, P. R. China 2 College of Animal Science and Technology, Northeast Agricultural University,

Harbin, Heilongjiang 150030, P. R. China 3Synergetic Innovation Center Of Food Safety and Nutrition, Northeast Agricultural

University, Harbin, 150030, P. R. China 4 Heilongjiang Research Center for Stem Cell Engineering, Harbin, Heilongjiang

150028, P. R. China

* Corresponding author. Tel.: +86-451-55190096

E-mail address: shidf@neau.edu.cn (D. Shi)

Figure S1. The schematic outline of the recombination strategy for constructing attenuated E. coli

O142: △STa. Panel A: Construction of the O142: △STa according to Gene Doctoring method and

λ-Red method. H1 and H2 refer to the homology extensions or regions. A1 and A2 refer to primers

for amplifying the KanR. Panel B: Schematic view of donor plasmids pDOC-C and pDOC-K.

Below is a linear representation of the pDOC plasmid, showing the I-SceI restriction sites, cloning

regions, the FLP recognition sites flanking the KanR in plasmids pDOC-C and pDOC-K. Panel C:

Schematic view of pACBSCE plasmid. I-SceI and the λ-Red gene are under the control of the

arabinose promoter and the repressor araC. Panel D: Schematic view of pCP20 plasmid. The FLP

recombinase gene is under the control of the PR promoter and the temperature-sensitive repressor

CIts857 of the phage λ. Panel E: Result of PCR reaction on E. coli O142, O142: Kan, and O142:

△STa using the primers A3 and A4. M: molecular size marker; N: PCR negative control; 1: PCR

product of E. coli O142; 2: PCR product of O142/pSTa donor; 3: PCR product of O142: Kan; 4:

PCR product of O142: △STa. Panel F: Suckling mice assay for identifying the toxicity of

attenuated E. coli O142: △STa. G/C ratios of ≥ 0.090 (indicated by a dotted line) are considered

as positive for STa enterotoxicity. Mean values are shown, and error bars represent standard

deviations. Panel G: ZYM-DIEC02 cells inoculated with supernatant of E. coli. a: cells inoculated

with supernatant of O142 showed significant cell death; b: cells inoculated with supernatant of

O142: △STa grew normally; c: cells non-treated as normal grew normally.

Figure S2. The schematic outline of the recombinant strategy for generating the double selection

platform. Panel A: Construction of the double selection platform O142(yaiT::PRPL-Kil) according

to Gene Doctoring method. The pKil-donor plasmid and the recombineering plasmid pACBSCE

are co-transformed into the recipient strain. Arabinose induction promotes expression of the λ-Red

gene products and I-SceI. I-SceI generates a linear DNA fragment form the pKil-donor plasmid

that is a substrate for recombination with the chromosome mediated by the λ-Red. Recombinants

are selected by the ability to survive and grow on LBKan supplemented with sucrose. Panel B: PCR

analysis of chromosomal DNA from double selection platform O142(yaiT::PRPL-Kil) by using

the primers A11 and A14. M: molecular size marker; N: PCR negative control; 1: PCR product of

E. coli O142: △ STa; 2: PCR product of O142: △ STa/pKil-donor; 3: PCR product of

O142(yaiT::PRPL-Kil). Panel C: Inversion screen test of the double selection platform. a:

O142(yaiT::PRPL-Kil) incubated at 43°C cannot grow on MacConkey agar plates; b:

O142(yaiT::PRPL-Kil) incubated at 30°C grown normally.

Figure S3. The schematic outline of the recombinant strategy for constructing the recombinant E.

coli O142(yaiT:: LT192-STa13) for oral vaccine candidate. Panel A: Construction of the LT192-

STa13 fusion gene. PCR primers A17 and A18 amplified the entire LT cassette including the native

LT promoter and terminator. Primers A18 paired with A19, A20 and A21 amplified the

STa13-6*His-terminator chimeric gene. Primers A15 and A16 mutated the LT gene for LT192.

Primers A21and A22 added a Gly-Pro-Gly-Pro linker and genetically fused the LT192 gene and the

STa13-6*His-terminator chimeric gene. Panel B: Construction of the recombinant E. coli

O142(yaiT:: LT192-STa13) according to Gene Doctoring method. The pL-S-donor plasmid and the

recombineering plasmid pACBSCE are co-transformed into the double selection platform.

Arabinose induction promotes expression of the λ-Red gene products and I-SceI. I-SceI cleaves

the pL-S-donor plasmid resulting in generation of the linear DNA fragment for λ-Red mediated

recombination to generate the recombinant E. coli O142(yaiT:: LT192-STa13). Panel C: PCR

reaction for the verifying of the recombinant E. coli strain ER-A by using the primers A24 and A25.

M: molecular size marker; N: PCR negative control; 1: PCR product of E. coli O142: △ STa; 2:

PCR product of O142(yaiT::PRPL-Kil); 3: PCR product of ER-A. Panel D: Detection of the

LT192-STa13 fusion protein in the Western blot assay. M: Protein marker; 1: E. coli O142: △ STa as

the negative control; 2: E. coli ER-A.

Figure S4. Feasibility of recombinant E. coli ER-A for oral vaccine candidate. Panel A: Suckling

mice assay for identifying the toxicity of ER-A. The toxicity of ER-A had eliminated or reduced.

Panel B: ZYM-DIEC02 cells inoculated with supernatant of E. coli. a: cells inoculated with

supernatant of 274-A showed significant cell death; b: cells inoculated with supernatant of O142

showed significant cell death; c: cells inoculated with supernatant of ER-A grew normally; d: cells

non-treated as normal grew normally. Panel C: The gastric acid tolerance of ER-A, from using the

plate method to enumerate the amounts of ER-A that survived in different pH of gastric acid. Panel

D: The intestinal juice tolerance of ER-A, from using the plate method to enumerate the amounts

of ER-A. Panel E: The bile tolerance of ER-A, from using the plate method to enumerate the

amounts of ER-A that survived in different concentrations of bile. Panel F: The daily appetite of

mice orally fed with ER-A. Panel G: The change of avoirdupois of mice orally fed with ER-A.

Panel H: The growth curve of ER-A. Bacterial growth was determinate by the number of CFU at

the time points indicated. Panel I: Electron microscope observations on the structure of fimbriae of

E. coli ER-A, O142: △ STa and O142 (magnification 30000 x). Panel J: Colonization efficacy of

ER-A in intestinal tracts of mice. Mean values are shown, and error bars represent standard

deviations.

Figure S5. In vitro neutralization assays on ZYM-DIEC02 cells. Panel A: Serum, intestinal mucus,

splenocyte lysate and mesenteric lymphocytes lysate from immunized mice showed neutralization

A

B

efficiency to STa toxin when compared with that from control mice. Panel B: Serum, intestinal

mucus, splenocyte lysate and mesenteric lymphocytes lysate from immunized mice showed

neutralization efficiency to LT toxin when compared with that from control mice. The ratios on

the figure were dilution gradient of toxins, the minimum inhibitory gradient of samples from the

immunized mice causing ZYM-DIEC02 cells to death greater than that of control mice.