Genome-wide screen of Salmonella genes expressed during infection in pigs, using in vivo expression technology

Abstract

Pigs are a food-producing species that readily carry Salmonella but, in the great majority of cases, do not show clinical signs of disease. Little is known about the functions required by Salmonella to be maintained in pigs. We have devised a recombinase-based promoter-trapping strategy to identify genes with elevated expression during pig infection with Salmonella enterica serovar Typhimurium. A total of 55 clones with in vivo-induced promoters were selected from a genomic library of approximately 10,000 random Salmonella DNA fragments fused to the recombinase cre, and the cloned DNA fragments were analyzed by sequencing. Thirty-one genes encoding proteins involved in bacterial adhesion and colonization (including bcfA, hscA, rffG, and yciR), virulence (metL), heat shock (hscA), and a sensor of a two-component regulator (hydH) were identified. Among the 55 clones, 19 were isolated from both the tonsils and the intestine, while 23 were identified only in the intestine and 13 only in tonsils. High temperature and increased osmolarity were identified as environmental signals that induced in vivo-expressed genes, suggesting possible signals for expression.

FIG. 1.

Identification of in vivo-induced genes. A genomic library of approximately 10,000 random Salmonella DNA fragments was fused to cre and preselected on kanamycin (Kan^r) to eliminate constitutive promoters from the population. The library was then administered to two pigs, and the intestinal contents were cultured on selective medium containing 5% sucrose (Suc^r), selecting for bacteria that lost the loxP cassette, along with the intervening sacB, due to the differential expression of cre. Each plasmid was reintroduced into the strain carrying the intact loxP cassette by P22 transduction; of 173 transductants, 154 remained kanamycin resistant. These were divided into groups of 20 to 35 each, and each group was used to infect two pigs. Sucrose-resistant Salmonella isolates were isolated from the ileum and the tonsils, and these were used to make pooled probes by PCR amplifying the cloned fragments of each. Probes were used in colony blots to determine which members of the input pool had reproducibly converted to sucrose resistance after animal infection, producing 55 promoter fragments that were expressed again in both of the pigs that had received the bacterial pool.

FIG. 2.

Effects of temperature on in vivo-induced gene fusions. Strains carrying a fusion of the indicated gene to lacZ were grown as standing overnight cultures in LB at 30°C (white bars), 37°C (gray bars), and 42°C (black bars). Triplicate cultures of each strain were assayed for lacZ expression using CRPG-enhanced β-galactosidase assays. Single asterisks show a significant increase (P ≤ 0.05) when the strain was grown at 42°C or 37°C compared to 30°C. Double asterisks show a significant increase (P ≤ 0.05) when the strain was grown at 42°C compared to 37°C. The β-galactosidase concentration was calculated as defined in Materials and Methods. Error bars show standard deviations. STM2689 is shown as a representative of fusions that were not induced under these conditions.

FIG. 3.

Effects of osmolarity on in vivo-induced gene fusions. Strains carrying a fusion of the indicated gene to lacZ were grown as standing overnight cultures in MOPS minimal medium (gray bars) or with the addition of 0.4 M NaCl (black bars). Triplicate cultures of each strain were assayed for lacZ expression in CRPG-enhanced β-galactosidase assays. All fusions shown, except for STM2689, produced a significant increase (P ≤ 0.05) in expression due to the addition of the NaCl. STM2689 is shown as representative of fusions that were not induced under these conditions. The β-galactosidase concentration was calculated as defined in Materials and Methods. Error bars show standard deviations.