Experimental strains The standard ETEC strain 10407 and a strain with the Tat knocked out were provided by the State Key Laboratory of Infectious Diseases Control and Prevention, National Institute for Communicable Disease Control and Prevention, Beijing, China.
Reagents and instruments Immobilized pH gradient (IPG) gel strips and protein quantitative kit were purchased from Amersham Pharmacia. The sequencing trypsin and protease inhibitor cocktail tables were purchased from Sigma, USA. PCR was performed using the PTC200 amplification system (MJ Research, USA). PROTEAN IEF CELL and Protean II XL cell (BioRad, USA), respectively were used for isoelectric focusing electrophoresis and vertical electrophoresis. 4700 MALDI-TOF-MS was used for mass spectrometry (Applied Biosystems, USA). Light microscope (Olympus Tokyo, Japan) as used for observing cell morphological changes.
Experimental animals: Male New Zealand white rabbits weighing 2–3 kg were purchased from the Institute of Laboratory Animal Sciences, CAMS & PUMC.
Identification of ETEC Tat deletion
Tat deletion was identified using PCR with deletion identification primers. The following forward and reverse primers were used for deletion identification: 5′-AAC GTA TAA TGC GGC TTT GTT-3′ and 5′-ATA TCA AAC ATC CTG TAC TCC-3′, respectively. In addition, morphological changes in the ETEC wild-type and Tat-deletion strains were observed under a light microscope.
Bacterial motility test
Sterilized liquid medium containing 0.3% agar was prepared, and an appropriate amount was added into a sterile U-shaped tube. Single colonies were inoculated into the medium for static culture after incubation at 37℃ overnight. The bacterial solution was diluted 1:50 in fresh medium for static culture at 37℃ for 3–4 h. Subsequently, 10 µL of the above bacterial solution was inoculated onto the liquid surfaces at both ends of the U-shaped tube, the tube was incubated at 37℃ for 4–6 h, and the results were observed.
Animal toxicity test
Healthy male rabbits were fasted for 24 h before operation and subjected to ligation of the intestinal segment in accordance with the routine method . Single colonies of ETEC10407 wild-type and Tat-deletion strains were inoculated in liquid medium and cultured overnight at 37℃. After ligation of the intestinal segment, 1 mL of the bacterial solution to be tested was injected into each animal using a sterile syringe. Rabbits were euthanized by embolization of the marginal ear veins at 24 h after operation. Intestinal segments were removed, cleaned, fixed in 40% formalin, embedded in paraffin, sectioned, stained with Hematoxylin and eosin (HE) and were observed for pathology under a light microscope.
Protein 2-DE and mass spectrometry
ETEC wild-type and Tat-deletion strain cultures were centrifuged and sonicated. The supernatant was collected by centrifugation to obtain bacterial protein samples, which were quantified using the Bradford method and subjected to 2-DE. Isoelectric focusing was performed in the following manner. Dry IPG gel strips (pH 4–7 and pH 3–10, 17 cm) were loaded in the rehydration buffer containing 600 µg of the sample.(50 V for 12–16 h, 250 V for 30 min, 1000 V for 1 h, and 10000 V for 11–12 h). SDS-PAGE was performed at a constant current of 10 mA for at least 10 h. After electrophoresis, the gel was stained with Coomassie brilliant blue G-250 and destained with 1% acetic acid.
Image information was digitized using a UMAX powerlook 2100XL scan (transmission scan with an optical resolution of 300bpi) and was analyzed using Pdquest (Bio-Rad). Differential proteins were detected and analyzed by mass spectrometry and the NCBI NR database was searched using a GPS workstation.