A Salmonella enterica serovar typhimurium translocated leucine-rich repeat effector protein inhibits NF-kappa B-dependent gene expression

Abstract

Nontyphoidal salmonellae are enteric pathogens that cause acute gastroenteritis and colonize the intestinal tract for prolonged periods. In the intestinal epithelia, these bacteria induce secretion of proinflammatory cytokines, such as interleukin-8 (IL-8), which leads to a profound inflammatory response through recruitment of polymorphonuclear leukocytes. Production of IL-8 induced by Salmonella spp. is due to the activation of the transcription factors nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1). This work demonstrates that Salmonella enterica serovar Typhimurium can downmodulate IL-8 production after invasion of intestinal epithelial cells. The Salmonella translocated effector proteins SspH1 and SptP participate in this process. SspH1 is a member of the bacterial LPX repeat protein family that localizes to the mammalian nucleus and inhibits NF-kappa B-dependent gene expression. A Shigella flexneri translocated effector, IpaH9.8, which has a similar structure and subcellular localization in mammalian cells, also inhibits NF-kappaB-dependent gene expression. We propose that suppression of inflammatory responses by intracellular S. enterica serovar Typhimurium, and perhaps Shigella flexneri, contributes to bacterial colonization of host tissues and pathogenesis.

FIG. 1.

SspH1 is localized to the nucleus in mammalian cells. Intestine-407 cells were infected with S. enterica serovar Typhimurium expressing SspH1-HA-HA (A) or SseJ-HA-HA (B). Cells were fixed, permeabilized, and immunostained for HA (red) and Salmonella LPS (green). Cell nuclei were visualized by staining with DAPI (blue). Raw-TT10 (C) and CHO-K1 (D) cells were transiently transfected with constructs expressing SspH1-HA, SlrP-HA, or SspH2-HA. HA-tagged proteins were detected by immunofluorescent staining (red), while DNA was stained with DAPI (blue).

FIG. 2.

SspH1 inhibits NF-κB-dependent reporter gene expression. CHO-K1 cells were transiently transfected with the indicated constructs: (A) a plasmid in which expression of firefly luciferase is under the control of the ELAM-1 promoter, a construct in which Renilla luciferase is expressed from the herpes simplex virus thymidine kinase promoter, vectors expressing mouse CD14 and the tetracycline transctivator, plus an empty, SspH1-expressing, or SspH1-nonexpressing vector (Reverse sspH1); (B and C) a plasmid in which expression of firefly luciferase is under the control of either ELAM-1 (B) or the Rous sarcoma virus promoter (C), a construct in which Renilla luciferase is expressed from the herpes simplex virus thymidine kinase promoter, vectors expressing mouse CD14 and the tetracycline transctivator, plus increasing concentrations of SspH1 expression vector (0, 2.5, 5, and 7.5 μg), while the amount of introduced DNA was kept constant by the addition of empty vector. After 24 h, the cells were incubated with E. coli LPS (solid bars) or fresh medium (open bars) for 5 h, and luciferase activity was measured. Firefly luciferase activity was normalized to the activity of Renilla luciferase. Data are represented as the mean ± standard deviation of triplicate samples.

FIG. 3.

The Shigella LPX repeat protein IpaH9.8 also inhibits NF-κB-dependent reporter gene expression. CHO-K1 cells were transiently transfected with pELAM-1-fluc, pRL-TK, pCDNA3.1/Zeo/mCD14, pNeo/Tak, and an empty vector or a construct expressing one of the following LPX repeat proteins: SspH1 (A and B), SlrP (A), SspH2 (A), or IpaH9.8 (B). After 24 h, the cells were incubated with E. coli LPS (solid bars) or fresh medium (open bars) for 5 h, and luciferase activity was measured. Firefly luciferase activity was normalized to the activity of Renilla luciferase. Data are represented as the mean ± standard deviation of triplicate samples. Each LPX repeat protein-expressing vector is diagramed to the right of its corresponding empty vector.

FIG. 4.

S. enterica serovar Typhimurium expressing SspH1 inhibits IL-8 production by Intestine-407 cells. Cells were infected with wild-type (CS401), ΔsspH1 (EM124), ΔsspH1 complemented with a plasmid expressing SspH1 (AH74), or ΔsptP (MJH2362) S. enterica serovar Typhimurium strains at a multiplicity of infection of 50. After 1 h, the cells were washed and incubated with medium containing gentamicin plus TNF-α (A) or only gentamicin (B) for another 5 h. Culture supernatants were collected, and the levels of secreted IL-8 were measured by ELISA. Data are represented as the mean ± standard deviation of quadruplicate samples.