Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli
Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli
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DOI：
10.1128/iai.74.4.2245-2258.2006
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发表时间：
2006-04-01
影响因子：
3.1
通讯作者：
Burkitt, M
中科院分区：
文献类型：
--
作者：
Fleckenstein, JM;Roy, KS;Burkitt, M
产肠毒素大肠杆菌(ETEC)仍然是世界范围内腹泻疾病的一个可怕的原因。目前，还没有针对ETEC提供广泛保护的疫苗。基于‘PhoA的自克隆突变系统TnPhoA.ts用于鉴定新的ETEC表面抗原，导致在原型株H10407的pCS1毒力质粒上发现ETEC两个伙伴分泌位点(EtpBAC)。EtpBAC的克隆和在重组大肠杆菌LMG194(PJY019)中的表达可分泌高相对分子质量(HMW)糖基化外源蛋白。这种糖蛋白EtpA呈现线性多肽序列，并预测与其他两个伙伴分泌基因座产生的HMW粘附素具有结构同源性。针对重组EtpA的抗体(抗rEtpA.6H)在ETEC H10407和LMG194(PJY019)的培养上清液中识别HMW蛋白，而在缺失92kb pCS1质粒的H10407-P菌株的培养上清液中不识别HMW蛋白，或等位基因的etpA突变体。EtpA突变体在体外缺乏与肠上皮细胞的黏附，抗rEtpA.6H抗体可抑制H10407与靶上皮细胞的结合。EtpB的克隆和在重组大肠杆菌中的表达足以产生粘附性。通过菌落杂交和EtpA免疫印迹对ETEC分离株的etpBAC基因进行筛选，表明EtpA是这些病原菌最常见的分泌抗原之一。综上所述，这些结果表明，新发现的ETEC双伴分泌基因座指导着一种高分子糖基化蛋白EtpA的分泌，该蛋白与假定的EtpB转运蛋白一起参与H10407与上皮细胞的黏附，从而扩大了潜在的ETEC毒力蛋白和候选疫苗的库。
Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A 'phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.