Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS)
Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS)
复制标题
DOI：
10.1046/j.1365-2958.2001.02593.x
复制
发表时间：
2001-09-01
影响因子：
3.6
通讯作者：
Curtiss, R
中科院分区：
文献类型：
--
作者：
Daigle, F;Graham, JE;Curtiss, R
伤寒沙门氏菌（S. typhi）是一种仅感染人类的病原体，可引发伤寒。由于该疾病的动物模型因伤寒沙门氏菌在其他宿主中缺乏毒力而受到严重限制，所以人们对伤寒沙门氏菌与宿主的相互作用了解相对较少。其他沙门氏菌血清型在动物模型中的毒力取决于这些细菌在宿主巨噬细胞内存活的能力。我们利用转录序列的选择性捕获技术（SCOTS）来鉴定伤寒沙门氏菌在人巨噬细胞内生长过程中表达的基因。这种阳性cDNA选择技术鉴定出28个不同的克隆，它们代表了先前已鉴定的以及新的、未表征的和假设的在细胞内表达的基因序列。利用SCOTS技术从细胞内细菌中获得了Vi荚膜抗原的转录本以及其产物参与应激反应和营养获取的基因。这些克隆中的大多数存在于鼠伤寒沙门氏菌基因组中，并且也在鼠巨噬细胞中表达。其中19个基因序列在伤寒沙门氏菌中被插入性破坏，与野生型亲本菌株相比，大多数由此产生的突变体在巨噬细胞内的存活水平较低。用野生型基因副本转化的突变菌株表现出与亲本菌株相似的巨噬细胞存活水平。
Salmonella enterica serovar Typhi (S. typhi) is a human-restricted pathogen which causes typhoid fever. Relatively little is known about S. typhi host interaction as animal models of this disease are severely limited by the lack of virulence of S. typhi in other hosts. The virulence of other Salmonella serovars in animal models is dependent on the abilities of these bacteria to survive within host macrophages. We have used selective capture of transcribed sequences (SCOTS) to identify S. typhi genes expressed during growth in human macrophages. This positive cDNA selection technique identified 28 distinct clones representing previously identified as well as novel, uncharacterized and hypothetical gene sequences that are expressed intracellularly. Transcripts for the Vi capsular antigen and genes whose products are involved in stress responses and nutrient acquisition were obtained from intracellular bacteria using SCOTS. Most of these clones are present in the S. typhimurium genome and are also expressed in murine macrophages. Nineteen of these gene sequences were disrupted insertionally in S. typhi, and most of the resulting mutants exhibited a lower level of survival within macrophages compared with the wild-type parent strain. Mutant strains, transformed with a copy of a wild-type gene, exhibited a macrophage survival level similar to that of the parental strain.