Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients
Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients
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DOI：
10.1128/iai.71.10.5461-5471.2003
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发表时间：
2003-10-01
影响因子：
3.1
通讯作者：
Rhee, JH
中科院分区：
文献类型：
--
作者：
Kim, YR;Lee, SE;Rhee, JH
病原菌的许多重要毒力基因在体内优先表达。本研究利用新近发展的体内诱导抗原技术（IVIAT）鉴定创伤弧菌体内诱导的基因。通过菌落印迹分析筛选创伤弧菌的表达文库，所述菌落印迹分析通过使用已经与体外表达的创伤弧菌全细胞和裂解物充分吸附的合并的恢复期血清进行。选择12个克隆，并分析插入DNA的序列。DNA序列与编码不同功能蛋白质的基因具有同源性：这些功能包括趋化性（一种甲基接受趋化蛋白），（一种含GGDEF的蛋白质和一种推定的丝氨酸/苏氨酸激酶），生物合成和代谢（PyrH、PurH和IlvC），分泌（TatB和质粒无色杆菌分泌[PAS]因子）、转录激活（IlvY和HlyU）和推定脂蛋白（YaeC）的活性。此外，一个确定的开放阅读框编码一个假设的蛋白质。构建了12个体内表达（ive）基因的同基因突变体，并进行了细胞毒性试验。细胞毒性活性的突变株，测定乳酸脱氢酶从HeLa细胞释放，几乎废除pyrH，purH，和hlyU突变体。小鼠腹腔注射50%致死量增加约10%。10-在这三个突变体中增加了50倍。PyrH和PurH似乎是体内生长所必需的。HlyU似乎是体内毒力表达的主要调节因子之一。如本研究所做的，成功鉴定了负责体内细菌毒力的基因，证明了IVIAT用于检测新毒力基因的有用性。
Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.