| 1. Background and ObjectiveEnterohaemorrhagic Escherichia coli （EHEC） O157:H7 is a new food-borne zoonotic intestinal bacterium, which can cause hemorrhagic colitis （HC）, haemolytic uraemic syndrome （HUS）, thrombotic thromobocytopenic purpura （TTP） and other diseases.In 1982, the American scholar Riley acquired EHEC O157:H7 from hemorrhagic diarrhea patients’ faeces for the first time. Thereafter, some sporadic cases and small outbreaks caused by EHEC O157:H7 continue to occur in the world. In 1996, from May to August, there was an outbreak of EHEC O157:H7 in Sakai city, Osaka, Japan. It involved more than 40 cities and counties, cumulative infected more than 9000 people, mostly for children aged 6-12. It killed 11 people, caused widespread concern and attention of the world. In 2000, there was an outbreak involving 5000 peoples caused by EHEC O157:H7 in Walkerton of the city Ontario, Canada.There were 5 people were killed. According to reports, it was caused by contamination of the water supply system in Walkerton.In 1986, Chinese isolated EHEC O157:H7 from hemorrhagic colitis patients, since then there were many small-scale outbreaks. From 1999 to 2000, there was an outbreak of EHEC O157:H7 in Jiangsu and Anhui province. It infected more than 20000 people and killed 177 people, lasted about 7 months. It was considered to be the largest outbreak, causing the largest number of deaths and continues the longest time as so far. Currently, EHEC O157:H7 outbreaks still occur, it has an upward trend and with strong pathogenicity and lethality. It is still a great threat to human health.The pathogenic role of EHEC O157:H7 is mainly reflected in two aspects:the secretion of Shiga-like toxin （Stx） and the adhesion and effacing lesions （A/E lesions）. The Shiga-like toxin has two types:Stxl and Stx2. It is encoded by sit gene in the prophage and able to cause VERO cells lesions, so it is called VERO toxin. Stx is an important pathogenic factor that causes HC and HUS. When EHEC O157:H7 bacteria come into the human intestines, it adheres to intestinal epithelial cells closely and makes intestinal epithelial cytoskeletal rearrangement, tight junctions between cells destroy, intestinal microvilli actin to dimerization, leading shrinkage and loss of microvilli, results to A/E lesions.The mainly virulence gene cause A/E lesions located on locus of enterocyte effacement （LEE） pathogenicity island. LEE pathogenicity island is comprised by five operon:LEE1 to LEE5. It encodes a variety of important virulence factors. EspF gene is located on the LEE4 ends and encodes secreted effector protein EspF （EHEC-secreted protein F）, which is the most extensive and functional role in the secreting effector proteins. Studies on Enteropathogenic Escherichia coli （EPEC） showed that bacteria injects EspF protein into the intestinal epithelial cells through type three secretion system （TTSS）, then EspF proteins can be combined with a variety of protein molecules in host cells and cause destruction of the host nucleolar, mitochondria, intermediate filaments and intercellular tight junctions. It also makes cell membrane reconstruction, cytoskeletal rearrangement, pedestal formation and results in apoptosis. It is sure that EspF protein is closely related to the mechanism of A/E lesions, so academics called it "Swiss Army knife" of A/E lesion pathogenic bacteria.Bioinformatics analysis shows that the N-terminal （l-73aa） of EspF protein in EHEC O157:H7 contains a secretion signal （1-20aa）, amitochondrial targeting signal （MTS） （l-24aa）, a nucleolar targeting domain （NTD） （21-74aa）. The C-terminal contain 4 PRRs, each PRR is highly homologous, contains an eukaryotic cell sorting nexin 9（SNX9） protein binding sites src homology 3 （SH3） motif, a neuronal wiskott-aldrich syndrome protein （N-WASP） binding domain, a possible actin binding motif （ABM）.The MTS site in EHEC O157:H7 EspF protein N-terminal is able to bind to host cell mitochondria. Research shows that if making the 16th leucine mutation to glutamate, the EspF protein can no longer bind to the mitochondria. If we knock out some sequences in N-terminal including MTS with suicide plasmid, the adhesion and induce cells apoptotic ability of bacteria decrease. The 4 PRRs in C-terminal contains SH3 binding motif PxxP, which could specifically binding the SH3 protein in the hosts, such as SNX9 protein. The SNX9 protein contains 3 regions SH3, PX and BAR, and SH3 is comprised with 50-60 amino acids. It mainly recognises the cell signaling proteins rich in proline sequence PxxP module, mediated protein- protein interactions. SNX9 protein is a regulator of cell membrane, can cause the formation of membrane tubules, induce membrane reconstruction and promote bacterial invasion.At present, the function of EspF protein of EPEC was researched deeply, but the study of EspF protein of EHEC is less. It’s not clear about the interactions between EspF and intestinal epithelial cells, the mechanism of apoptosis that EspF induced and the function of the domain of EspF. In the early work, our group constructed EHEC O157:H7 EspF N-terminal deletion mutant and proved that the N-terminal of EspF protein in EHEC O157:H7 can targeted bind to the host mitochondria and induce apoptosis. But the EspF protein C-terminal PRR region can bind to the protein with SH3 domain, such as SNX9, they form a complex and large protein-protein interaction networks, and there is a link with the cell signal transduction pathway and apoptosis. So how these protein start or mediated apoptosis? Whether hemorrhagic enteritis is related to these proteins? A further study is needed. According to the structure of EspF protein, we construct 3 EHEC O157:H7 EDL933w gene deletion mutant strains with λ Red recombination system:espF gene deletion mutant strains （EHEC O157:H7 EDL933w △espF）, espF gene N-terminal deletion mutant strains （EHEC O157:H7 EDL933w △espF-N） and espF gene C-terminal deletion mutant strains （EHEC O157:H7 EDL933w △espF-C）. While we construct the compensation strains, compare the differences of biological, adhesion and ability of induce host cells apoptosis between them. Preliminary studying the function of each domain of EHEC O157:H7 EspF protein.2. Methods2.1 Construction of Escherichia coli O157:H7 espF-domain-deleted strains and compensation strains（1） Construction of espF-domain-deleted strainsAccording to the gene sequences of espF in GenBank （Accession NO. NC02655）, we designed two pairs of primers containing homology arms:H1K1, H2K2 and H3K3, H4K4. H1K1 and H2K2 are used to construct espF knockout targeting fragment, H3K3 and H2K2 are used to construct espF-N knockout targeting fragment, are used to construct espF knockout targeting fragment are used to construct espF-C knockout targeting fragment. The 5’end of these four primers is homologous arms and 3’end is kana gene. We use PKD4 plasmid containing kanamycin-resistant gene as a template and PCR amplification, construct knockout targeting fragment of espF, espF-N and espF-C. Then we electroporate the targeting fragment into EHEC O157:H7 EDL933w strain containing pKD46 plasmid, induce pKD46 plasmid expressing recombinase with L-arabinose and the targeting fragment recombines with target gene. Then we screen positive bacteria with kanamycin and design two pairs of primers to checkout if recombination occurs.（2） Construction of compensation strainsAccording to the gene sequences of espF in GenBank （Accession NO. NC02655）, we design two pairs of primers:Fc, Rn and Fn, Rc. Fc and Rn are used to PCR espF gene fragments, Fn and Rn are used to PCR espF-N gene fragments, Fc and Rc are used to PCR espF-C gene fragments. We construct pGEM-espF, pGEM-espF-N, pGEM-espF-C vector, then transform it into DH5 a and screen positive bacteria with ampicillin. Testing positive bacteria by PCR and isolate the bacteria DH5α pGEM-espF, DH5 a pGEM-espF-N, DH5 a pGEM-espF-C. Electroporate the vector into knockout bacteria △espF, △espF-N, AespF-C.2.2 Biological characteristics compare of wild and mutant strainsWild and mutant strains make Gram staining according to the steps, observing it under microscope. Under the same culture conditions, wild and mutant strains are incubated and measured OD600 values in 0,1.5,3,4,6,8,10,12,14,16,20h. Then drawing the growth curve draw.2.3 Adhesion test（1） Giemsa staining bacterial adhesionEHEC O157:H7 wild and mutant strains was adjusted to the same concentration and inject the Lovo cells for 2h. Staining according to the steps of Giemsa staining and observing strain adhesion to Lovo cell under microscope.（2） Computing bacterial adhesion rateEHEC O157:H7 wild strain, △espF, △espF-N,△espF-C and DH5 a are incubated overnight, resuspended to antibiotic-free medium （DMEM,10% fetal bovine serum）, the concentration of bacteria is 2× 107/mL. Lovo cells are cultured in 12-well plates, grow to monolayer cells （about 2 × 105 cells/well）. Inoculating bacteria into 12-well plates which Lovo cell cultured, each well 2 X 107 bacteria （cell multiplicity of infection 100:1）. Gently mix plates and put it into incubator,37 ℃, 5% CO2, incubate for 1.5-2 h. Each bacterial inoculate four wells. Washing plates for 3 times with PBS after incubation cells and adding 150 μL Triton X-100 of 0.5%. After inoculating for 8 min, adding 100 μ L PBS, repeat pipetting samples, gradient dilution and spreading on the agar plates. After 24h, do agar plate count and calculate adhesion rate （adhesion rate= the number of bacteria adhere to cells/total bacteria added into cells well × 1000‰）. Analyzing data by One Way ANOVA and comparing the difference adhesion rate between strains.3. Results3.1 Constructed 3 EHEC O157:H7 EDL933w espF gene deletion strainsThe targeting fragments are successfully constructed by PCR technology and electroporated into EHEC O157:H7 EDL933w containing plasmid pKD46. Positive strains were screened by kanamycin plates and identified by espF internal primers. 471bp fragment can be seen in mutant strains, no fragments were found in wild strains. After sequencing, it is sure that EHEC O157:H7 gene espF （747bp）, espF-N（219bp） and espF-C （528bp） are completely deleted.The compensating fragments are successfully constructed by PCR technology and bound to vector pGEM-T easy. Positive strains were screened by ampicillin plates and identified by universal primers M13. Incubating bacteria and obtaining the plasmid, electroporating the plasmid into knochout strain △ espF, △espF-N, △espF-C.3.2 Biological characteristics compare of wild and mutant strainsIn Gram staining, wild strain and mutants are gram-negative, corynebacterium parvum. According to the growth curve, growth curve trend of wild strain and mutant strains are basically the same.3.3 Adhesion testIn Giemsa staining, there are more wild strains adhesion to cells than mutant strains. Adhesion Number:WT strain is higher than mutant strains. The adhesive bacteria are coated plates and counted, calculate the rate of bacterial adhesion, ONE WAY ANOVA analysis showed that the adhesion between the strain rate comparison P<0.05, statistically significant. The average adherence rate of wild strains is 13.879 ±3.578‰, mutant strain △espFis 6.401+1.519‰,, mutant strain △espF-N is 5.402 ±3.212‰, mutant strain △espF-C is 4.795±1.950‰.4. Conclusion4.1 EHEC O157:H7 EDL933w espF deletion mutant strains are successfully constructed:△ espF, △ espF-N, △ espF-C. The compensating strains are also successfully constructed. These strains make a foundation for the further research of EHEC O157:H7 EspF protein.4.2 espF gene or espF domain deletion do not affect the form and growth rhythm of EHEC O157:H7.4.3 espF gene or espF domain deletion reduce the ability of adhesion to intestinal epithelial cells, adhesion:WT strain is higher than mutant strains, mutant strains are higher than DH5 a. |
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