Faucher Sébastien P, Porwollik Steffen, Dozois Charles M, McClelland Michael, Daigle France
Department of Microbiology and Immunology, University of Montreal, C.P. 6128 Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7.
Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1906-11. doi: 10.1073/pnas.0509183103. Epub 2006 Jan 27.
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 peptides, 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.
通过转录序列选择性捕获获得的cDNA是一种复杂混合物,可与微阵列结合使用,以确定病原体在感染期间的全局基因表达。我们使用这种方法来研究伤寒热的病原体——伤寒沙门氏菌在人类巨噬细胞内表达的基因。获得并比较了伤寒沙门氏菌在体外和巨噬细胞内不同时间点生长时的全局表达谱。发现已知的毒力因子,如SPI-1和SPI-2编码的III型分泌系统,在沙门氏菌感染期间如预期那样表达,这验证了我们的数据。巨噬细胞内的伤寒沙门氏菌显示出编码对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。编码鞭毛装置、趋化性和铁转运系统的基因在体内下调。许多对应于功能未知基因的cDNA在人类巨噬细胞内上调,对于未来阐明这种人类特异性病原体的细胞内生活方式的研究而言,考虑这些基因将很重要。实时定量PCR与微阵列结果一致。转录序列选择性捕获和微阵列的联合使用是确定体内细菌转录组的有效方法,可用于研究其他细菌病原体的转录谱,而无需从宿主中回收许多纳克细菌mRNA,也无需将感染复数增加到超过自然所见的水平。