Salmonella pathogenicity island 2 is expressed prior to penetrating the intestine

Abstract

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes disease in mice that resembles human typhoid. Typhoid pathogenesis consists of distinct phases in the intestine and a subsequent systemic phase in which bacteria replicate in macrophages of the liver and spleen. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is a major virulence factor contributing to the systemic phase of typhoid pathogenesis. Understanding how pathogens regulate virulence mechanisms in response to the environment, including different host tissues, is key to our understanding of pathogenesis. A recombinase-based in vivo expression technology system was developed to assess SPI-2 expression during murine typhoid. SPI-2 expression was detectable at very early times in bacteria that were resident in the lumen of the ileum and was independent of active bacterial invasion of the epithelium. We also provide direct evidence for the regulation of SPI-2 by the Salmonella transcription factors ompR and ssrB in vivo. Together these results demonstrate that SPI-2 expression precedes penetration of the intestinal epithelium. This induction of expression precedes any documented SPI-2-dependent phases of typhoid and may be involved in preparing Salmonella to successfully resist the antimicrobial environment encountered within macrophages.

Figure 1. Expression from P_sseA during Growth of Salmonella in Inducing and Non-Inducing Culture Media

(A–C) The wild-type Salmonella RIVET strain (NB25) was inoculated into LPM (inducing) and LB (non-inducing) media at 1/100 of the culture volume, and cultures were incubated with shaking at 37 °C. At the indicated time points, samples were taken for measurement of culture OD₆₀₀ (unpublished data), colony-forming units per millilitre (unpublished data), β-galactosidase activity, percent resolution, and native SseB levels. The results shown for percent resolution (A) and β-galactosidase activity (B) are the mean ± standard error of the mean from three independent experiments. The levels of SseB associated with bacteria were detected by immunoblotting (C). Protein loading was normalized to culture OD₆₀₀ and was confirmed by immunoblotting for the abundantly expressed cytoplasmic protein DnaK. (D) Wild-type (NB25), ssrB ⁻ (NB7), and ompR ⁻ (NB15) were grown in LPM medium and percent resolution was determined from the cultures at 10 h.

Figure 2. Expression from P_sseA during Infection of Mammalian Cells In Vitro

The human epithelial cell line HeLa, murine macrophage cell line RAW264.7, and bone-marrow-derived DCs were infected with wild-type (NB25), ssrB ⁻ (NB7), and ompR ⁻ (NB15) RIVET strains as described in Materials and Methods. At 1 h post-infection intracellular bacteria were recovered and the percent resolution was determined.

Figure 3. Kinetics of SPI-2 Expression during Murine Typhoid

(A) Mice were infected with the wild-type P_sseA RIVET strain (NB25) as described in Materials and Methods. At the indicated times following inoculation, the ileal loops, mesenteric lymph nodes, spleen, and liver were taken and assessed for expression from P_sseA by determining the percent resolution. The results shown are the mean ± standard error of the mean from three independent experiments. (B) Mice were infected with the wild-type (NB25), ssrB ⁻ (NB7), and ompR ⁻ (NB15) P_sseA RIVET strains, and at 15 min following inoculation, the ileal loop was removed and homogenized, and the percent resolution of the infecting Salmonella was determined. (C) Mice were infected with the wild-type P_spiC (NB33) and P_ssaG (NB31) RIVET strains for 15 and 30 min, the ileum was removed and homogenized, and the percentage resolution determined. The results shown are the mean ± standard error of the mean for three independent experiments.

Figure 4. Salmonella Induces Expression of SPI-2 in the Lumen of the Ileum

(A) Mouse ileal loops were infected with either wild-type or invA ⁻ P_sseA RIVET strains for 15 min before the loops were removed and homogenized, and the percent resolution was determined. (B) Mouse ileal loops were infected with wild-type Salmonella for 15 min before being removed, fixed, stained, and analyzed by confocal microscopy with an oil immersion 40× 1.3 N.A. objective. A representative image is displayed showing host cell nuclei in blue, actin in green, and Salmonella in red.