A Salmonella enterica serovar Typhi plasmid induces rapid and massive apoptosis in infected macrophages

Abstract

pR(ST98) is a chimeric plasmid isolated from Salmonella enterica serovar Typhi (S. typhi) that mediates the functions of drug resistance and virulence. Previously, we reported that Salmonella plasmid virulence (spv) genes were present in S. typhi. In our current study, we investigated whether plasmid pR(ST98) exhibits significant cytotoxicity in macrophages. pR(ST98) was transferred into the avirulent Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create the transconjugant pR(ST98)/RIA. The standard S. typhimurium virulent strain SR-11, which carries a 100-kb virulence plasmid, was used as a positive control. The bacterial strains were incubated with a murine macrophage-like cell line (J774A.1) in vitro. Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling, and the survival of Salmonella strains in J774A.1 cells was determined. Results showed that macrophages infected with strain pR(ST98)/RIA displayed greater levels of apoptosis than those infected with RIA and that pR(ST98 )may increase bacterial survival in macrophages. Further studies showed that the pR(ST98)-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pR(ST98 )may activate caspase-9 and then caspase-3. The research data indicate that the virulence of bacteria that contain the pR(ST98) plasmid is enhanced; the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.

Figure 1

Plasmid electrophoresis profiles and the identification of conjugal transfer of pR_ST98. Lane 1, E. coli V517, plasmid size marker (54.4, 7.3, 5.6, 5.2, 4.0, 3.0, 2.7 and 2.1 kb); lane 2, S. flexneri 24570, plasmid size marker (159.6, 4.0 and 3.0 kb); lane 3, E. coli K12W1485, plasmid-free; lane 4, multidrug resistant S. typhi carrying a 150-kb plasmid (pR_ST98); lane 5, E. coli K12W1485 receiving pR_ST98, the plasmid donor in the second conjugal transfer step; lane 6, S. typhimurium strain SR-11 carrying a 100-kb virulence plasmid; lane 7, avirulent S. typhimurium strain RIA carrying a 136.8-kb R plasmid; and lane 8, transconjugant pR_ST98/RIA carrying two plasmids (136.8 and 150 kb). E. coli, Escherichia coli; S. flexneri, Shigella flexneri; S. typhi, Salmonella enterica serovar Typhi; S. typhimurium, Salmonella enterica serovar Typhimurium.

Figure 2

PCR amplification of Salmonella plasmid virulence (spv) genes. The open reading frames of spvR (894 bp) and spvB (1776 bp) were amplified. M, Axygen 100 bp DNA ladder; lanes 1, 4: transconjugant S. typhimurium strain pR_ST98/RIA; lane 2, 5: S. typhimurium strain SR-11; lane 3, 6: avirulent S. typhimurium strain RIA. S. typhimurium, Salmonella enterica serovar Typhimurium.

Figure 3

Transmission electron micrographs of J774A.1 cells infected with S. typhimurium. (a) Uninfected macrophages show normal cellular morphology, including intact plasma and nuclear membranes. (b–e) J774A.1 macrophages at 3 h post-infection. (b) RIA-infected macrophages. (c) pR_ST98/RIA-infected macrophages. (d) and (e) SR-11-infected macrophages. Black arrows denoted intracellular growth of Salmonella strains in macrophages. Macrophages infected with pR_ST98/RIA or SR-11 display characteristic features of apoptosis, including condensed and marginated nuclear chromatin (arrowhead), membrane blebbing (white arrows) and cytoplasmic vacuolation. However, cells infected with RIA appeared normal, although they contained intracellular bacteria. (f–h) J774A.1 macrophages at 24 h post-infection. Many of the J774A.1 cells infected with pR_ST98/RIA (g) and SR-11 (h) underwent necrosis; the ultrastructure was significantly worse than that in macrophages infected with RIA (f). S. typhimurium, Salmonella enterica serovar Typhimurium.

Figure 4

Assessment of J774A.1 cells with flow cytometry after annexin-V/propidium iodide labeling. (a, e) uninfected cells; (b, f) J774A.1 cells infected with S. typhimurium strain RIA at 3 and 24 h post-infection, respectively. (c, g) J774A.1 cells infected with S. typhimurium strain pR_ST98/RIA at 3 and 24 h post-infection, respectively. (d, h) J774A.1 cells infected with S. typhimurium SR-11 at 3 and 24 h post-infection, respectively. The results are presented as the percentage of cells that were viable (Ann V^- PI^-, represented in the lower left quadrant of the dot plot) and early apoptotic (Ann V⁺ PI^-, the lower right quadrant). Fig. 4B shows the mean percentage of S. typhimurium-infected J774A.1 cells that underwent apoptosis from three separate experiments. In comparison with uninfected and S. typhimurium RIA-infected cells, pR_ST98/RIA-infected macrophages displayed an increase in apoptosis similar to that detected in SR-11-infected cells (*P<0.05; **P<0.01; n=9). Ann V, annexin-V; PI, propidium iodide; S. typhimurium, Salmonella enterica serovar Typhimurium.

Figure 5

Measurement of mitochondrial membrane potential of J774A.1 cells. (a) The percentage of mitochondria membrane potential (Δψ_m) decrease in J774A.1 cells. At 1 h post-infection, S. typhimurium strain pR_ST98/RIA resulted in a higher number of J774A.1 with decreased mitochondrial Δψ_m than RIA, and a time-dependent decrease in mitochondrial Δψ_m was observed. (b, c) Fluorescent JC-1 reaction examination of J774A.1 cells undergoing mitochondria Δψ_m decrease detected with a laser scanning cytometer (×200) and laser scanning confocal microscope, respectively. The corner panels are higher magnifications of the boxed areas. S. typhimurium, Salmonella enterica serovar Typhimurium.

Figure 6

Caspase-9 and caspase-3 activities in culture supernatants of J774A.1 cells infected with S. typhimurium. Cultures were harvested at 5 h post-infection. Values are expressed as means±standard deviation of seven different observations. *Significantly different (P<0.01) from uninfected control and RIA-infected cells. S. typhimurium, Salmonella enterica serovar Typhimurium.

Figure 7

Bacterial survival in J774A.1 cells. Bacterial Log CFU per 10⁵ macrophage cells (y axis) and time after addition of amikacin (x axis) are indicated. The results are presented as the mean±standard error of the mean. CFU, colony-forming unit.