Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS).
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.
Daigle
F
F
Department of Biology, Washington University, One Brookings Drive, Campus Box 1137, St Louis, MO 63130, USA.
Graham
J E
JE
Curtiss
R
R
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D012485:Salmonella typhi / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014435:Typhoid Fever / Q000382:microbiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D018076:DNA, Complementary