Identification of attenuated Yersinia pseudotuberculosis strains and characterization of an orogastric infection in BALB/c mice on day 5 postinfection by signature-tagged mutagenesis

Abstract

Yersinia pseudotuberculosis localizes to the distal ileum, cecum, and proximal colon of the gastrointestinal tract after oral infection. Using signature-tagged mutagenesis, we isolated 13 Y. pseudotuberculosis mutants that failed to survive in the cecum of mice after orogastric inoculation. Twelve of these mutants were also attenuated for replication in the spleen after intraperitoneal infection, whereas one strain, mutated the gene encoding invasin, replicated as well as wild-type bacteria in the spleen. Several mutations were in operons encoding components of the type III secretion system, including components involved in translocating Yop proteins into host cells. This indicates that one or more Yops may be necessary for survival in the gastrointestinal tract. Three mutants were defective in O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type Y. pseudotuberculosis. Several other mutations were in genes that had not previously been associated with growth in a host, including cls, ksgA, and sufl. In addition, using Y. pseudotuberculosis strains marked with signature tags, we counted the number of different bacterial clones that were present in the cecum, mesenteric lymph nodes, and spleen 5 days postinfection. We find barriers in the host animal that limit the number of bacteria that succeed in reaching and/or replicating in the mesenteric lymph nodes and spleen after breaching the gut mucosa.

FIG. 1

Comparison of number of YPIIIpIB71 CFU recovered from the cecum, PP, and MLN of mice infected with a 3:1 ratio of WT Y. pseudotuberculosis to the Yop-deficient strain YPIIIpIB71 (mixed) with the number of CFU from mice infected with only YPIIIpIB71 at doses of 3 × 10⁸ (alone-low) and 3 × 10⁹ (alone-high). In the mixed infection experiment, a total of 3 × 10⁹ bacteria were used, of which 7.5 × 10⁸ were YPIIIpIB71. Four mice were infected with the mixed inoculum, and five mice were infected with the low and high doses of YPIIIpIB71. Mice were sacrificed 5 days postinfection.

FIG. 2

Phenotypes of ST strains with cells in culture. (A) Percentage of ST strains recovered after bacteria were grown at 26°C in 2xYT containing 5 mM CaCl₂ and exposed to HeLa cells at a multiplicity of infection of 10:1. After a 30-min infection, gentamicin was added to the medium (first box in each pair) for an additional 90 min, the cells were washed and lysed with 1% Triton X-100, and the bacteria were plated. In a side-by-side control, gentamicin was not added to the medium cells were lysed with a final concentration of 1% Triton X-100 and bacteria were plated (second box in each pair). All experiments were done in triplicate, and each strain was tested at least two times in both assays. (B) Percentage of gentamicin-resistant bacteria of various ST strains after infection of HeLa cells with bacteria pregrown at 37°C for 2 h. HeLa cells were infected as described above and in Materials and Methods. (C) Percentage of RAW264.7 macrophage cell death due to infection with Y. pseudotuberculosis relative to complete lysis of the macrophages. The bacteria were pregrown at 37°C for 2 h, the cells were infected for 2 h, gentamicin was added to the medium, and culture supernatants were assayed 7 h after the addition of gentamicin. All experiments were performed in triplicate and repeated twice. The strain labeled lcrR is YPIIIpIB71, which does not secrete any Yops.