Host microarray analysis reveals a role for the Salmonella response regulator phoP in human macrophage cell death

Abstract

Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoPTn10 mutant strain. Both wild-type and phoPTn10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoPTn10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.

Figure 1

Wild-type and phoP∷Tn10 mutant Salmonella replicate in PMA-treated U-937s. At 2 (gray bars) and 24 (black bars) hr after infection, cells were lysed, and bacterial cfu were plated. WT, wild-type; phoP, phoP∷Tn10 mutant; spiA, spiA∷Tn5 mutant.

Figure 2

Comparison of array and Northern data depicted as levels of mRNA in infected relative to uninfected macrophages. RNA from one of the array experiments was processed for Northern and probed with labeled PIM-1 or SSI-3 cDNA. (A) PIM-1 array (Left) and Northern (Right) data. (B) SSI-3 array (Left) and Northern (Right) data. The Northern data were normalized to a glycerol-3-phosphate dehydrogenase 1 (GPD-1) Northern. Data are expressed as the geometric mean of the bacterial-infected vs. uninfected macrophage ratios from the 2- to 4-hr time points. WT, wild-type-infected macrophages; phoP, phoP∷Tn10-infected macrophages.

Figure 3

A phoP∷Tn10 mutant killed fewer U-937 macrophages than wild-type S. typhimurium by 4 hr after infection. (A) Suspended and adherent cells were harvested and processed for FACS. The percentage of TUNEL-positive events is shown. (B) Adherent U-937s were TUNEL and DAPI stained for fluorescence microscopy. U, uninfected; WT, wild-type; phoP, phoP∷Tn10 mutant; orgA, orgA∷Tn10 mutant.

Figure 4

A phoP∷Tn10 mutant killed fewer U-937 macrophages than wild-type S. typhimurium by 20 or 24 hr after infection. (A) Suspended and adherent cells were harvested and processed for FACS 20 hr after infection. The percentage of TUNEL-positive events is shown. (B) LDH release was measured 24 hr after infection. Results are presented as the percent cell death, where 100% is the amount of LDH in uninfected lysed cells. lysed, uninfected lysed; WT, wild-type; phoP, phoP∷Tn10 mutant; spiA, spiA∷Tn5 mutant.

Figure 5

A phoP∷ Tn10 mutant strain is defective for killing human PBMCs. Adherent PBMCs were TUNEL and DAPI stained for fluorescence microscopy 4 hr after infection. U, uninfected; WT, wild-type; phoP, phoP∷Tn10 mutant; orgA, orgA∷Tn10 mutant.