Identiﬁcation and Characterization of a K88- and CS31A-Like Operon of a Rabbit Enteropathogenic Escherichia coli Strain Which Encodes Fimbriae Involved in the Colonization of Rabbit Intestine-猫眼课题宝

编码参与兔肠道定植的菌毛的兔致病性大肠杆菌菌株的 K88 和 CS31A 样操纵子的鉴定和表征

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J. Girardeau;M. Vartanian;J. Ollier;M. Contrepois

附着-消失性病变的启动是兔致病性大肠杆菌 (REPEC) 感染的特征，需要细菌粘附在肠上皮上。这种粘附在体外通过这些大肠杆菌菌株对各种类型的真核细胞的亲和力反映出来。产生了REPEC 83/39 (O15:H 2 )的Tn pho A突变体，其失去了对HEp-2上皮细胞、豚鼠回肠刷状缘和小鼠红细胞的粘附能力。对 95 kb 质粒（称为 REPEC 粘附质粒的 pRAP）内失活转座子插入周围区域的 DNA 测序揭示了该区域与编码 K88 和 CS31A 菌毛粘附素的产肠毒素大肠杆菌操纵子的结构基因以及来自 REPEC B10 (O103:H2) 的 afr 2 粘附素基因之间的广泛同源性。对 ral 操纵子（用于 REPEC 粘附位点）的 7 个基因进行了测序，包括三个假定的次要菌毛亚基基因（ ralC 、 ralF 和 ralH ）、一个主要菌毛亚基基因（ ralG ）、一个功能未知的基因（ ralI ）和两个菌毛亚基伴侣基因（ ralD 和 ralE ）。当经口接种给刚断奶的兔子时，在 ralE 基因中插入 Tn pho A 的突变体表现出定植能力降低了 10 倍，与所有 5 只感染野生型亲本菌株的兔子相比，10 只兔子中只有 1 只分泌细菌（P 5 0.002）。由突变菌株引起的腹泻疾病的严重程度也降低了。用超免疫抗 83/39 抗血清对吸附有 ralE 突变体的菌株 83/39 的表面蛋白提取物进行蛋白质印迹，显示出 32-kDa 的蛋白，而两种非贴壁突变体的蛋白提取物中不存在该蛋白。通过免疫金标记检测，吸附的抗血清也与菌株 83/39 的表面结合，但不与非贴壁突变体结合。这些结果表明，REPEC 83/39 的 ral 操纵子含有生物合成细菌毛所必需的基因，细菌毛负责细菌在体外的粘附，并在细菌在兔子的定植中发挥作用，从而对兔子产生毒力。假定的主要菌毛亚基是一种蛋白质，观察到的分子大小约为 32 kDa，组装后似乎会形成围绕细菌的菌毛胶囊，类似于 CS31A 所描述的情况。然后用稀释液洗涤五次，用 PBS 洗涤五次，然后用 1% 钼酸铵对样品进行负染色。使用 Philips 400 透射电子显微镜在 60 kV 的加速电压下观察染色的制剂。兔子的细菌定植。为了确定 ral 操纵子在体内的作用，给 5 至 6 周大的新西兰白兔口服接种菌株 83/39（5 只兔子）或菌株 83/39/23（10 只兔子）。对于这些研究，细菌在 PAB 中生长，然后洗涤并重悬于 PBS 中。接种前，兔子通过胃管接受 2 ml 5%（重量/体积）过滤灭菌的碳酸氢钠。 15 分钟后，以相同方式施用 2 ml 含有 6 3 10 6 CFU 的适当细菌悬浮液。连续三周每天监测受感染兔子的体重减轻和腹泻迹象，例如稀便和后肢弄脏。观察期结束时，静脉注射戊巴比妥钠处死腹泻兔。通过在前 14 天每天培养直肠拭子来半定量测定细菌排泄量，此时野生型和突变株的相对定殖能力存在明显差异。将普通无菌棉签插入肛门约 2 厘米。将拭子上的粪便物质在 0.5 ml PBS 中乳化，并铺在补充有利福平 (50 m g/ml) 的麦康凯琼脂上，以阻止所有非攻击细菌的生长。这种计数方法的可靠性在其他研究中得到证实，这些研究表明，以这种方式获得的 CFU 数量反映了尸检时测量的每 0.01 g 结肠内容物的实际 CFU (1)。定期检查从感染突变菌株的兔子中分离出的细菌对卡那霉素的耐药性，以确保没有与感染野生型细菌的兔子发生交叉污染。当连续3天检测到细菌排泄时，就认为兔子有定植。核苷酸序列登录号。 GenBank 加入

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Esche- richia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reﬂected in vitro by the afﬁnity of these E. coli strains for various types of eukaryotic cells. Tn pho A mutants of REPEC 83/39 (O15:H 2 ) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transpo- son insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A ﬁmbrial adhesins and the genes for the afr 2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor ﬁmbrial subunit genes ( ralC , ralF , and ralH ), a major ﬁmbrial subunit gene ( ralG ), a gene of unknown function ( ralI ), and genes for two ﬁmbrial subunit chaperones ( ralD and ralE ), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a Tn pho A insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain ( P 5 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of ﬁne ﬁmbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major ﬁmbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of ﬁmbriae surrounding the bacterium similar to that described for CS31A. ther washes with diluent and ﬁve with PBS, the sample was negatively stained with 1% ammonium molybdate. Stained preparations were viewed with a Philips 400 transmission electron microscope at an accelerating voltage of 60 kV. Bacterial colonization of rabbits. To establish a role for the ral operon in vivo, 5- to 6-week-old New Zealand White rabbits were inoculated perorally with either strain 83/39 (5 rabbits) or strain 83/39/23 (10 rabbits). For these studies, bacteria were grown in PAB and then washed and resuspended in PBS. Prior to inoculation, rabbits received 2 ml of 5% (wt/vol) ﬁlter-sterilized sodium bicar-bonate via a gastric tube. After 15 min, 2 ml of the appropriate bacterial sus- pension containing 6 3 10 6 CFU was administered in the same manner. Infected rabbits were monitored daily for 3 weeks for weight loss and evidence of diarrhea such as loose stools and soiling of the hind limbs. At the end of the observation period, diarrheic rabbits were killed by an intravenous injection of sodium pentobarbitone. Bacterial excretion was determined semiquantitatively by culturing rectal swabs daily for the ﬁrst 14 days, by which time an obvious difference in the comparative colonizing abilities of the wild-type and mutant strains was established. Plain sterile cotton-tipped swabs were inserted approx- imately 2 cm through the anus. Fecal material on the swabs was emulsiﬁed in 0.5 ml of PBS and plated on MacConkey agar supplemented with rifampin (50 m g/ml), which precluded growth of all nonchallenge bacteria. The reliability of this counting method was established in other studies in which it was shown that the numbers of CFU obtained in this way reﬂected the actual CFU per 0.01 g of colon contents measured at autopsy (1). Bacteria isolated from rabbits infected with the mutant strain were checked periodically for resistance to kanamycin to ensure that no cross-contamination from rabbits infected with the wild-type bacteria had occurred. A rabbit was considered to be colonized when bacterial excretion was detected on 3 consecutive days. Nucleotide sequence accession number. The GenBank accession

Identiﬁcation and Characterization of a K88- and CS31A-Like Operon of a Rabbit Enteropathogenic Escherichia coli Strain Which Encodes Fimbriae Involved in the Colonization of Rabbit Intestine

编码参与兔肠道定植的菌毛的兔致病性大肠杆菌菌株的 K88 和 CS31A 样操纵子的鉴定和表征

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