| Avian pathogenic Escherichia coli can cause a variety of diseases which generally known as avian colibacillosis. The varied clinical symptoms and complex pathological changes resuted in a great economic losses in the poultry industry.The LuxS/AI-2 type quorum sensing system exists in gram-negative and gram-positive bacteria which involves the production of autoinducer-2 (AI-2). The bacterial receptors sense and combine the AI-2 signaling molecules, thus the expression of related intracellular genes were further induced to regulate their biological characteristics eventually. The uptake of AI-2 also involves the activated methionine cycle (AMC). Two pathways of AMC exist in bacteria, which consists of pfs/luxS and sahH metabolic pathways. The regulation mechanism of AMC was still unknown in APEC.In this study, the differences on biological characteristics were investigated through constructing different AMC pathways in APEC, which will benefit for development of the pathogenesis, diagnosis, treatment, prevention and control of APEC.(1) Cloning, expression and activity detection of LuxS, Pfs and SahH in vitroIn order to verify the feasibility of building sahH metabolic pathway in DE17 (Serotype 02). The prokaryotic expression vectors named pET28a-luxS, pET28a-pfs and pET28a-sahH were constructed. The recombinant proteins Pfs, LuxS and SahH were expressed in BL21 (DE3). The 400 μmol/L HCY can be produced after catalysis of SAH by the recombinant proteins LuxS and Pfs, while 32.7 mol/L HCY by recombinant protein SahH.(2) Construction of different pathways of AMC in APECFor evluation different pathways of AMC in APEC, the pfs mutant strain DE17Δpfs was constructed, and the pfs/luxS methionine metabolism pathway was blocked. The complementary vector pSTV-sahH was constructed by sahH of Pseudomonas aeruginosa. Then the plasmid pSTV-sahH was transferred to DE17 or DE17Δpfs, and the complementary strains sahH-DE17 and sahH-DE17Apfs were constructed, respectively. The sahH-DE17 exists pfs/luxS and sahH metabolism pathways, while the sahH-DE17Δpfs only exists sahH metabolism pathway.The results of real-time PCR showed that the level of transcription of sahH is high in sahH-DE17 and sahH-DE17Δpfs. The results of AI-2 activity assays confirmed that DE17Δpfs and sahH-DE17Δpfs could not produce AI-2. Compared with DE17, the AI-2 in sahH-DE17 was significantly inhibited, the sixth hour was the most significant. The results will provide a foundation for studying AMC pathways in APEC.(3) Effects on the biological characteristics of APEC in different AMC pathwaysFor investigation of the biological characteristics of APEC in different AMC pathways, the following of biological characteristics was evulated:1) The growth curve. Compaird with DE17, the growth rate of DE17Δpfs is consistent with sahH-DE17Δpfs, both of which were significantly slower than DE17.2) The determination of the LD50. Compared with wild-type strain DE17 (LD50 is 1.24x103CFU), the virulence of DE17Δpfs (LD50 is 8.06×105 CFU) reduced to 650 folds, the sahH-DE17Apfs (LD50 is 6.49x104 CFU) reduced to 52.3 folds, while sahH-DE17 (LD50 is 6.49×104 CFU) showed no significant difference.3) The bacterial loads in vivo. Compared with DE17 (The bacterial loads in blood, liver, spleen and kidney is 2.1×106 CFU/mL,1.7×107 CFU/g,2.3×108 CFU/g and 4.1×108 CFU/g, respectively), the DE17Δpfs (The bacterial loads in blood, liver, spleen and kidney is 1.6×102 CFU/mL,7.5×102 CFU/g,1.1 ×104 CFU/g and 1.4×104 CFU/g, respectively) reduced significantly (P< 0.001), and also in the sahH-DE17Δpfs (P< 0.01), while the sahH-DE17 (The bacterial loads in blood, liver, spleen and kidney is 3.1 ×102CFU/mL, 1.7×107 CFU/g,2.3×108 CFU/g and 4.1 ×108CFU/g, respectively) showed no significant difference, except in liver (P< 0.001).4) The results of adhesion and invasion to DF-1 cell. The adhesional ability of DE17 is 1.1 ×107 CFU/well, and the invasional ability is 1.4×104 CFU/well, while the DE17Δpfs is 6.9x106 CFU/well and 7.6x103 CFU/well, respectively. Compard with DE17, the DE17Apfs is significantly decreased (P< 0.01). Furthermore, the adhesional ability of sahH-DE17 is 9.8x106 CFU/well, and the invasional ability was 8.4x103 CFU/well. Compard with DE17, the adhesional ability changes no significantly difference, but the invasional ability significantly decreased (P< 0.01). Moreover, compard with DE17, the adhesional and invasional ability of sahH-DE17Δpfs changes without significantly difference.The present study indicates that the pfs/luxS metabolic pathway plays an important role in regulation of the pathogenicity in APEC, which will benefit for study the pathogenic mechanism and control of APEC based on AMC. |
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