QseC mediates Salmonella enterica serovar typhimurium virulence in vitro and in vivo

Abstract

The autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE) interkingdom signaling system mediates chemical communication between bacteria and their mammalian hosts. The three signals are sensed by the QseC histidine kinase (HK) sensor. Salmonella enterica serovar Typhimurium is a pathogen that uses HKs to sense its environment and regulate virulence. Salmonella serovar Typhimurium invades epithelial cells and survives within macrophages. Invasion of epithelial cells is mediated by the type III secretion system (T3SS) encoded in Salmonella pathogenicity island 1 (SPI-1), while macrophage survival and systemic disease are mediated by the T3SS encoded in SPI-2. Here we show that QseC plays an important role in Salmonella serovar Typhimurium pathogenicity. A qseC mutant was impaired in flagellar motility, in invasion of epithelial cells, and in survival within macrophages and was attenuated for systemic infection in 129x1/SvJ mice. QseC acts globally, regulating expression of genes within SPI-1 and SPI-2 in vitro and in vivo (during infection of mice). Additionally, dopamine beta-hydroxylase knockout (Dbh(-)(/)(-)) mice that do not produce Epi or NE showed different susceptibility to Salmonella serovar Typhimurium infection than wild-type mice. These data suggest that the AI-3/Epi/NE signaling system is a key factor during Salmonella serovar Typhimurium pathogenesis in vitro and in vivo. Elucidation of the role of this interkingdom signaling system in Salmonella serovar Typhimurium should contribute to a better understanding of the complex interplay between the pathogen and the host during infection.

FIG. 1.

QseC regulation of Salmonella serovar Typhimurium's flagella in vitro. (A) Motility assay performed in plates containing LB medium with 0.3% agar after 8 h. (B) Sizes of motility halos after 8 h. (C) Motility assay performed with the WT and ΔqseC strains using LB medium without NE after 8 h. (D) Motility assay performed with the WT and ΔqseC strains in LB medium with 50 μM NE after 8 h. (E) Sizes of motility halos after norepinephrine (NE) addition at 8 h. (F) Transcription of flhDC in the WT strain and the ΔqseC mutant in LB medium with and without 50 μM norepinephrine (Nore). (G) Transcription of flagellar genes (flhDC, fliA, and motA) in WT, ΔqseC, and complemented strains in motility agar. Statistical significance was determined by Student's t test based on comparison with the expression in the WT strain (*, P < 0.001). qseC+, complemented strain.

FIG. 2.

Involvement of QseC in Salmonella serovar Typhimurium invasion of HeLa cells and intramacrophage survival. (A) Invasion of HeLa epithelial cells by WT, ΔqseC, and complemented strains. (B) Intramacrophage survival (J774 macrophages) of WT, ΔqseC, and complemented strains. Statistical significance was determined by Student's t test based on comparison with the WT strain (*, P < 0.001). qseC+, complemented strain.

FIG. 3.

QseC regulates transcriptional expression of SPI-1 and sifA in vitro. (A) Transcription of SPI-1 genes in WT, ΔqseC, and complemented strains in LB medium. (B) Transcription of sifA in WT, ΔqseC, and complemented strains in N-minimal medium. (C) Transcription of invF (SPI-1), sopB (SPI-1), and sifA in WT and ΔqseC strains in LB medium in the absence and presence of 50 μM NE. In all cases expression levels were compared to WT expression levels without NE. In addition, a second analysis of the expression of invF was performed by comparing the expression in the WT strain with NE and the expression in the ΔqseC mutant with NE, as indicated. Statistical significance was determined by Student's t test (*, P < 0.001). Nore, norepinephrine; qseC+, complemented strain.

FIG. 4.

QseC's role in murine systemic infection. (A) Survival curves for 129x1/SvJ mice infected intraperitoneally (IP) with WT Salmonella serovar Typhimurium, the ΔqseC isogenic mutant, and E. coli K-12 as control (P < 0.0005, as determined using Student's t test and comparing the qseC mutant to the WT at every time point on the survival curve until 5 days postinfection). (B) Organ loads of WT Salmonella serovar Typhimurium and the ΔqseC isogenic mutant during systemic dissemination in 129x1/SvJ mice at 20 h postinfection (hpi) (P < 0.0005). (C) Competitive index (CI) for the qseC mutant and WT strain SL1344 in 129x1/SvJ mice 20 h postinfection (ratio of ΔqseC systemic dissemination to WT systemic dissemination). Statistical significance was determined by Student's t test (P < 0.02).

FIG. 5.

QseC regulates transcriptional expression in vivo of SPI-1, sifA, and SPI-3. (A) Transcription of sipA and sopB in spleens and livers harvested from 129x1/SvJ mice 20 h after IP infection with the WT or the ΔqseC mutant. (B) Transcription of sifA and mgtB in spleens and livers harvested from 129x1/SvJ mice 20 h after IP infection with the WT or the ΔqseC mutant. Statistical significance was determined by Student's t test (*, P < 0.001). Sp, spleens; Lv, livers.

FIG. 6.

Comparison of transcriptional expression in WT strain SL1344 in vitro (LB broth) and in vivo (spleens and livers). All statistical and expression analyses were performed by comparing expression levels to LB broth expression levels. Statistical significance was determined by Student's t test (*, P < 0.001).

FIG. 7.

Salmonella serovar Typhimurium infection in Dbh^−/− knockout mice. (A) Survival plots for Dbh^+/− and Dbh^−/− mice infected IP with WT Salmonella serovar Typhimurium strain SL1344. (B) Survival plots for Dbh^+/− and Dbh^−/− mice infected IP with the ΔqseC mutant. Statistical significance was determined by Student's t test.

FIG. 8.

Differential transcriptional expression of SPI-1 and SPI-3 in Dbh^−/− mutant mice. Transcription of sipA, sopB, and mgtB in spleens and livers harvested from WT and Dbh^−/− mice 20 h after IP infection with the WT and the ΔqseC mutant. Statistical significance was based on a comparison of expression levels to expression levels in WT Salmonella serovar Typhimurium in Dbh^+/+ mice and was determined by Student's t test (*, P < 0.001). Sp, spleens; Lv, livers; hpi, hours postinfection.