The potassium transporter Trk and external potassium modulate Salmonella enterica protein secretion and virulence

Abstract

Potassium (K(+)) is the most abundant intracellular cation and is essential for many physiological functions of all living organisms; however, its role in the pathogenesis of human pathogens is not well understood. In this study, we characterized the functions of the bacterial Trk K(+) transport system and external K(+) in the pathogenesis of Salmonella enterica, a major food-borne bacterial pathogen. Here we report that Trk is important for Salmonella to invade and grow inside epithelial cells. It is also necessary for the full virulence of Salmonella in an animal infection model. Analysis of proteins of Salmonella indicated that Trk is involved in the expression and secretion of effector proteins of the type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) that were previously shown to be necessary for Salmonella invasion. In addition to the role of the Trk transporter in the pathogenesis of Salmonella, we discovered that external K(+) modulates the pathogenic properties of Salmonella by increasing the expression and secretion of effector proteins of the SPI1-encoded TTSS and by enhancing epithelial cell invasion. Our studies demonstrated that K(+) is actively involved in the pathogenesis of Salmonella and indicated that Salmonella may take advantage of the high K(+) content inside host cells and in the intestinal fluid during diarrhea to become more virulent.

FIG. 1.

Growth of the ΔtrkA mutant of Salmonella with a high concentration of potassium. Wild-type strain SE2472, the ΔtrkA mutant, and the complemented mutant (ΔtrkA-comp) were cultured in minimal K⁺ medium supplemented with 50 mM KCl. Bacterial concentrations were determined by plating. At least three experiments were performed, and the results of a representative experiment performed in triplicate are shown. The error bars indicate standard deviations.

FIG. 2.

Epithelial cell invasion and intracellular growth of the wild-type strain and the ΔtrkA mutant of Salmonella. (A) Invasion of HeLa cells. The ratio of the number of intracellular bacteria to the number of input bacteria was determined for wild-type strain SE2472 (WT), the ΔtrkA mutant, and the complemented mutant (ΔtrkA-comp). The ratio for wild-type strain SE2472 at 1 h postinfection was arbitrarily defined as 100%, and the ratios for other samples were expressed as relative values. (B) Growth inside HeLa cells. Intracellular wild-type strain SE2472, ΔtrkA mutant, and complemented mutant (ΔtrkA-comp) bacteria were quantified, and the results were compared to the initial number of bacteria at zero time. The increase was calculated by dividing the number of intracellular bacteria at 4 or 8 h by the number of intracellular bacteria at zero time. The data are the averages of three experiments performed in triplicate. The error bars indicate standard deviations.

FIG. 3.

Expression and secretion of selected effector proteins of the SPI1-encoded TTSS by the wild-type strain and ΔtrkA mutant of Salmonella. Epitope-tagged strains SipA(HF), SipC(HF), and SopB(HF) with the wild-type (WT) or ΔtrkA mutant allele and the ΔtrkA mutant transformed with plasmid pRB3-trkA (ΔtrkA-comp) were cultured in LB broth. The bacterial lysates or cultural supernatants were analyzed to determine the levels of SipA, SipC, and SopB in the tagged strains. (A) Western blot analysis of the SipA levels in whole-cell lysates. (B) Western blot analysis of the levels of SipA, SipC, and SopB in culture supernatants. The trkA allele in each sample is indicated above the lane. The effector proteins analyzed in the blots are indicated on the right.

FIG. 4.

Effects of external potassium and sodium chloride on the invasion of HeLa cells by the wild-type strain and ΔtrkA mutant of Salmonella. Salmonella wild-type strain SE2472 (WT) or the ΔtrkA mutant was cultured overnight in LB broth or LB broth supplemented with 50 or 100 mM KCl (A) or 50 or 100 mM NaCl (B) and used to infect HeLa cells. Intracellular Salmonella was quantified 1 h after infection, and the ratios of the number of intracellular bacteria to the number of input bacteria (expressed as percentages) were determined. The ratio for wild-type strain SE2472 grown without a supplement was arbitrarily defined as 100%, and the ratios for the other samples were expressed as relative values. The data are the averages of three experiments performed in triplicate. The error bars indicate standard deviations.

FIG. 5.

Comparison of the effects of external potassium and sodium chloride on the expression and secretion of selected effector proteins of the SPI1-encoded TTSS by the wild-type strain (WT) and the ΔtrkA mutant of Salmonella. Epitope-tagged strains SipA(HF), SipC(HF), and SopB(HF) with the wild-type trkA or ΔtrkA mutant allele were cultured in LB broth supplemented with 50 or 100 mM KCl or 50 or 100 mM NaCl (indicated above the lanes). The bacterial lysates or culture supernatants were analyzed to determine the levels of SipA, SipC, and SopB in the strains. (A) Western blot analysis of the SipA levels in whole-cell lysates. (B) Western blot analysis of the levels of SipA, SipC, and SopB in culture supernatants. The effector proteins analyzed in the blots are indicated on the right.