Hemagglutination by pilus antigen 987P of enterotoxigenic Escherichia coli

The hemagglutination (HA) by pilus antigen 987P of an enterotoxigenic Escherichia coli strain 987 was examined using fresh and glutaraldehyde (GA)-fixed erythrocytes (RBC) of various animals. Only when GA-fixed RBC was employed, a strain 987 exhibited striking HA activities. This was also demonstrated by using latex heads sensitized with the 987P antigen. The 987P-specific antiserum inhibited HA of strain 987 and 987P sensitized latex beads against GA-fixed RBC. We concluded that HA of strain 987 against GA-fixed RBC was specifically associated with the presence of 987P pilus antigen but do not exclude a possibility that adhesin is distinct from pili antigen.     

987P fimbriae from porcine enterotoxigenic Escherichia coli: Characterization, N-terminal amino acid sequence and immunization with purified antigen

Abstract The 987P fimbrial antigen was purified from a spontaneous overproducing variant. The protein was characterized with respect to M _r, amino acid sequence and partial covalent structure. The purified protein was used for vaccination trials, and excellent protection of piglets upon challenge with 987P positive enterotoxigenic strains was seen.     

Characterization of fimbriae extracts from porcine enterotoxigenic Escherichia coli strains carrying F6 (987P) antigen.

Fimbrial extracts from porcine enterotoxigenic Escherichia coli (ETEC) strains carrying F6 (987P) intestinal colonization factor antigen wereobtained using the thermal shock method. The extracts were analyzed by SDSPAGE and immunoblotting using different fimbriae-specific antisera. Two major protein bands with molecular masses of 17.5 and 21.9 kDa were detected. The 21.9-kDa band was identified as the major subunit of F6 fimbrial antigen in strains of serogroups O9 and O141. The 17.5-kDa band was associated with porcine strains of serogroups O9 and O20.     

Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat

The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (mu). Under aerobic growth conditions fimbriae production increased with higher mu values till mu = 0.40 h-1 and decreased again at mu values close to mu max (0.48 h-1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to mu max (0.16 h-1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of mu. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+ cell and a slower reduction in the percentage of P+ cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+ cells and a constant amount of fimbriae per P+ cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+----P- switch between two steady states was 2.7 x 10(-2). In batch culture the frequency of the P(-)----P+ switch was minimally 2.9 x 10(-2). The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.     

Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli

The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32-35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P.     

大肠杆菌K99菌毛fan操纵子的克隆、表达及活性

[目的]在体外克隆和表达猪产肠毒素大肠杆菌(ETEC) K99菌毛操纵子fan结构基因,并检测重组菌毛的相关生物学活性.[方法]以猪源分离的表达K99菌毛ETEC C83907株制取模板,成功PCR扩增出编码K99菌毛的fan操纵子,约5.7 kb.将fan操纵子克隆人表达质粒载体pBR322,筛选出含正确阳性质粒的重组菌.进一步将上述的重组质粒DNA转化至不含任何菌毛的大肠杆菌SE5000株,同时将空载体pBR322质粒转化入SE5000构建阴性对照菌株.[结果]该重组菌能与鼠抗K99菌毛单克隆抗体发生明显的凝集反应,与新生仔猪小肠上皮细胞刷状缘BBV分子有强烈凝集反应.电镜观察到上述重组菌表面大量表达K99菌毛,用热抽提法提纯其表达的K99菌毛,并经SDS-PAGE电泳和考马斯亮蓝染色,可以得到分子量约为18.5kDa的主要蛋白条带.纯化菌毛免疫小鼠后制备出高效价的鼠抗血清,能与携带K99菌毛的C83907、C83914、C83260野生株发生强烈的凝集反应,而与携带其他菌毛的ETEC不反应.玻板凝集试验和Western blot结果表明:体外表达的K99菌毛具有和野生K99菌毛相同的抗原性.用表达K99菌毛的重组菌进行HeLa细胞体外黏附试验和黏附抑制实验,结果表明:重组菌和野生菌株一样具有较强的粘附性,而且用重组菌毛制备的鼠抗血清能有效地抑制上述重组菌或野生菌株对细胞系的黏附结合.[讨论]本研究为进一步研究K99菌毛生物学作用建立了良好的实验平台.     

Regulators of the Bacillus subtilis cydABCD operon: identification of a negative regulator, CcpA, and a positive regulator, ResD

The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions -58 and -107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3'-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions -4 to -33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.     

Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene,a positive regulator of nif gene expression

We report the isolation, mutational analysis and the nucleotide sequence of the Rhizobium leguminosarum bv. phaseoli nifA gene. Comparison of the deduced amino acid sequence with other NifA sequences indicated the presence of the conserved central activator and the C-terminal DNA-binding domains. Nodules elicited by a R. leguminosarum bv. phaseoli nifA mutant were symbiotically ineffective. The expression of a nifA-gusA fusion was shown to be independent on the oxygen status of the cell. We cloned the three nifH copies of R. leguminosarum bv. phaseoli and determined the nucleotide sequence of their promoter regions. The expression of nifH-gusA fusions is induced under microaerobic conditions and is dependent on the presence of NifA.Abbreviations bp base pair(s) - kb kilobase(s) - ORF open reading frame     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

Characterization of the Escherichia coli gcvR gene encoding a negative regulator of gcv expression.

The Escherichia coli glycine cleavage enzyme system catalyzes the cleavage of glycine, generating CO2, NH3, and a one-carbon unit. Expression of the operon encoding this enzyme system (gcv) is induced in the presence of glycine and repressed in the presence of purines. In this study, a mutant with high-level constitutive expression of a gcvT-lacZ gene fusion was isolated. The mutation in this strain was designated gcvR1 and was mapped to min 53.3 on the E. coli chromosome. A single-copy plasmid carrying the wild-type gcvR gene complemented the mutation, restoring normal regulation of a gcvT-lacZ fusion, while a multicopy plasmid carrying gcvR led to superrepression under all growth conditions. Negative regulation of a gcvT-lacZ fusion by GcvR was shown to require GcvA, a LysR family protein known to both activate gcv in the presence of glycine and repress gcv in the presence of purines. Models explaining how GcvR and GcvA might interact to regulate gcv expression are proposed.     

Cloning of regulator genes controlling fimbrial production by enterotoxigenic Escherichia coli

Sequences regulating production of fimbriae were cloned from two enterotoxigenic Escherichia coli strains. One cloned region, from E. coli 0.25.H42, controlled expression of coli surface-associated (CS) antigen 4, whereas the function of the other, from E. coli 0167.H5, was unclear. Both regulators were related to the cfaD gene that controls expression of colonization factor antigen I (CFA/I) although low stringency conditions were required to show significant hybridization between cfaD and the regulatory fragment from E. coli 0167. The cloned regulatory genes promoted expression of CFA/I, CS1, CS2 and CS4 antigens but the levels of production in the presence of the 0167 regulator were lower than those promoted by the CS4 regulator or cfaD.