Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences
Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences
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DOI：
10.1073/pnas.0509183103
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发表时间：
2006-02-07
影响因子：
11.1
通讯作者：
Daigle, F
中科院分区：
文献类型：
--
作者：
Faucher, SP;Porwollik, S;Daigle, F
通过选择性捕获转录序列获得的 cDNA 是一种复杂的混合物，可与微阵列结合使用来确定病原体在感染过程中的整体基因表达。我们使用这种方法来研究人类巨噬细胞内伤寒沙门氏菌（伤寒的病原体）表达的基因。获得并比较了不同时间点体外和巨噬细胞内生长的伤寒菌的整体表达谱。已知的毒力因子，例如 SPI-1 和 SPI-2 编码的 III 型分泌系统，被发现在沙门氏菌感染期间按照预测表达，这验证了我们的数据。巨噬细胞内的伤寒杆菌表现出编码抗菌肽抗性的基因表达增加，利用乙醛酸旁路进行脂肪酸利用，并且不诱导 SOS 反应或氧化应激反应。编码鞭毛器、趋化性和铁转运系统的基因在体内下调。许多与功能未知的基因相对应的 cDNA 在人类巨噬细胞内上调，对于未来研究阐明这种人类特异性病原体的细胞内生活方式非常重要。实时定量PCR与微阵列结果一致。选择性捕获转录序列和微阵列的结合使用是确定体内细菌转录组的有效方法，并且可用于研究其他细菌病原体的转录谱，而不需要从宿主中回收许多纳克的细菌mRNA，并且不会增加超出自然界中可见的感染复数。
The cDNA obtained by selective capture of transcribed sequences is a complex mixture that can be used in conjunction with microarrays to determine global gene expression by a pathogen during infection. We used this method to study genes expressed by Salmonella enterica serovar Typhi, the etiological agent of typhoid fever, within human macrophages. Global expression profiles of Typhi grown in vitro and within macrophages at different time points were obtained and compared. Known virulence factors, such as the SPI-1- and SPI-2-encoded type III secretion systems, were found to be expressed as predicted during infection by Salmonella, which validated our data. Typhi inside macrophages showed increased expression of genes encoding resistance to antimicrobial pepticles, used the glyoxylate bypass for fatty acid utilization, and did not induce the SOS response or the oxidative stress response. Genes coding for the flagellar apparatus, chemotaxis, and iron transport systems were down-regulated in vivo. Many cDNAs corresponding to genes with unknown functions were up-regulated inside human macrophages and will be important to consider for future studies to elucidate the intracellular lifestyle of this human-specific pathogen. Real-time quantitative PCR was consistent with the microarray results. The combined use of selective capture of transcribed sequences and microarrays is an effective way to determine the bacterial transcriptome in vivo and could be used to investigate transcriptional profiles of other bacterial pathogens without the need to recover many nanograms of bacterial mRNA from host and without increasing the multiplicity of infection beyond what is seen in nature.