Microbial gene expression elucidated by selective capture of transcribed sequences (SCOTS)

Selective Capture ofSalmonella entericaSerovar Typhi Genes Expressed in Macrophages That Are Absent from theSalmonella entericaSerovar Typhimurium Genome

“…As survival within macrophages is an essential step for Salmonella pathogenesis (10), we have used selective capture of transcribed sequences (SCOTS) (6,14) to identify genes expressed by serovar Typhi within macrophages. In order to obtain sequences unique to serovar Typhi (i.e., absent from the serovar Typhimurium genome), we have used an additional strain-specific enrichment step, previously described to identify in vivo-expressed pathogen-specific genes from avian-pathogenic Escherichia coli (8).…”

“…In order to obtain sequences unique to serovar Typhi (i.e., absent from the serovar Typhimurium genome), we have used an additional strain-specific enrichment step, previously described to identify in vivo-expressed pathogen-specific genes from avian-pathogenic Escherichia coli (8). The human macrophage-like cells, derived from the monocyte cell line THP-1 (ATCC TIB-202), were infected with serovar Typhi ISP1820 as described previously (5,6). The infected monolayers were lysed 2 h postinfection by the addition of TRIzol (Invitrogen), and RNA was prepared according to the manufacturer's instructions.…”

“…The infected monolayers were lysed 2 h postinfection by the addition of TRIzol (Invitrogen), and RNA was prepared according to the manufacturer's instructions. A 5-g sample of RNA was isolated from infected macrophages or under other growth conditions, treated with RNase-free DNase, and reverse transcribed by random priming (6,11). Three rounds of bacterial cDNA capture were done as described previously (5,6,14).…”

Novel AttenuatedSalmonella entericaSerovar Choleraesuis Strains as Live Vaccine Candidates Generated by Signature-Tagged Mutagenesis

“…Several methods for screening and identifying bacterial genes involved in conferring the ability to penetrate, colonize, and propagate in host tissues have been developed; these methods include in vivo expression technology (5,44), differential fluorescence induction (69), selective capture of transcribed sequences (11), in vivo induced antigen technology (25), substrate hybridization (55), and signature-tagged mutagenesis (STM) (29). These strategies, particularly STM, led to the identification of numerous attenuated mutants in S. enterica subsp.…”

Une bactérie au pays des biopuces : étude du transcriptome deSalmonelladans des macrophages infectés

“…capture of transcribed sequences) permet l'identification de gènes bactériens exprimés dans les cellules de l'hôte par clonage et séquençage des transcrits [1]. Nous avons récem-ment réussi à combiner les qualités de ces deux approches [2].…”