Objective To analyze the sequences of two component signaling system PhoP/PhoQ encoding genes of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics and to determine the correlation between the PhoQ/PhoP and the resistance. Methods The segments of entire pimQ and phoP genes of P. aeruginosa were obtained by PCR and then sequenced after T-A cloning. Two prokaryotic expression systems of phoQ and phoP genes were constructed and the target recombinant expres-sion products rPhoQ and rPhoP were extracted by Ni-NTA chromatography. Rabbits were intracutaneoualy immunized with rPhoQ and rPhoP to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ genes of aminloglycoside antibiotics-resistant P. aeruginosa strains were knocked out by using Red recombination system, and phoQ mutants were identified by PCR plus sequencing and Western blot assay. Tube dilution method was applied to determine MIC values of wild and mutant strains of P. aeruginosa to four different aminoglycoside antibiotics. Results In comparison with the corresponding sequences in GenBank, the similarities of nueleotide and putative amino acid sequences of the cloned phop and phoQ genes were 98.7%-99.6% and 98.7%-100% , and 98.4%-99.8% and 99.1%-100%, respec-tively. Both rPhoQ and rPhoP were successfully expressed using pET-42a and E. coil BL21 DE3 system, and their rabbit antisera with 1 : 4 and 1 : 8 double immunodiffusion titers were also obtained. The deletion of phoQ genes and absence of the products in the two phoQ mutants were confirmed by PCR, sequencing and West-ern blot assay. MIC values of the four different aminoglycoside antibiotics to the two mutants were 1/512-1/2048 as those of their wild strains. Conclusion PboQ/PhoP is a sequence conserved two component sig-naling system of P. aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics.

plasmid can be used to study the pathogenic mechanism of target gene products of L.interrogans.

Objective To determine the effects of Che A and CheY proteins of Campylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization in vivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector and expression strain, respectively, prokaryotic expression systems of cheA and cheY genes of C. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the target recombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG and rCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript- II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-out mutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotactic ability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting the bacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY after DOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. The difference of colonization ability between cheA- mutant and wild-type of C. jejuni in mice were checked and then compared. Results The constructed prokaryotic expression systems could efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed the cheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotactic ability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis of wild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levels of CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). The colonization ability in murine jejunum of cheA- mutant was also lower than that of the wild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system (Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activated by dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro and colonization in vivo of C. jejuni, and this protein can be used as a target for developing novel anti- C. jejuni drugs.

Objective To clone mcp1, mcp2 and mop3 genes that encoding methyl-accepting chemotaxis proteins(MCP) of Campy/obacter jejuni for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of the microbe for determining chemotaxis-inducing substances and to understand relationship among MCPs and chemotactic inducers. Methods The segments of mep1, mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning. Prokaryofic expression systems of the genes were subsequently constructed. SDS-PAGE pins Bin-Rod Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1, rMCP2 and rMCP3, and Ni-NTA affinity chromatography was performed to purify the rMCPs. Rabbits were immunized with each of the three rMCPs to obtain antisera. Immunodiffusion assay was performed to measure the titers of antisera. IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography, and IgG F(ab')2 were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography. Chemotactic model in vitro of C. jejuni based on HAP( hard-agar plus) method was established to determine the chemo-taxis-inducing effect of eight candidate substances. Chemotaxis inhibition test based on IgG F(ab')2 bloc-king was applied to determine the function and diversity of MCPs. Results The segments with expected si-zes amplified from mcp1, racp2 and mcp3 genes were obtained by PCRs, and their nucleotide and putative amino acid sequences were completely same as the reported. The constructed prokaryotic systems could effi-ciently express rMCPs with the yields about 10% of the total bacterial proteins. Immunization with rMCP1, MCP2 and rMCP3 enables the rabbits to produce specific antibody. All the antisera had 1: 4 immunodiffusion titers. Both bovine bile and sodium deoxycholate(DOC) were able to induce ehemotactie movement of C. je-juni in a dosage-dependent manner ( P < 0.05 ). When MCP1 and MCP2 were blocked with their IgG F(ab')2, the ehemotaetic ability of C. jejurd were remarkably decreased (P <0. 05). However, MCP3 blocking did not affect the chemotaxis ( P > 0.05 ). Conclusion The C. jejuni MCPs are successfully ex-pressed in this study. Bovine bile and DOC are the inducers for chemotaxis of C. jejuni, and MCP1 and MCP2 are involved in the process of ehemotaxis iadueed by DOC.

In this study ,we aimed to understand the sequence characteristics ,transmembrane structures ,line B cell epitopes present in the OMP18 from Campylobacter jejuni ,and provide candidate antigens for the antibody detection and vac-cine development .NCBI/Blast ,TMHMM Server V2 and DNA Star softwares were used for the OMP18 sequence analysis . Based on the ELISA ,the whole bacterial antibody IgG of Campylobacter jejuni was used for the identification of the predicted line B cell epitopes .The OMP18 gene was found conserved in different Campylobacter jejuni strains .The OMP18 was predic-ted to be located on the outer surface of the bacteria .And three line B cell epitopes were determined to be present in the OMP18 protein .As a conclusion ,the OMP18 protein was confirmed to be an important outer membrane protein ;three line B cell epitopes were identified in the OMP18 ,which could be further used for Campylobacter jejuni antibody detection and vaccine development .

Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.

OBJECTIVETo construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization.

METHODSThe plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice.

RESULTSThe fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05).

CONCLUSIONThe function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

Animals ; Bacterial Proteins ; genetics ; Campylobacter jejuni ; genetics ; pathogenicity ; Chemotaxis ; genetics ; Gene Knockout Techniques ; Jejunum ; microbiology ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C