speG Is Required for Intracellular Replication of Salmonella in Various Human Cells and Affects Its Polyamine Metabolism and Global Transcriptomes

Abstract

The speG gene has been reported to regulate polyamine metabolism in Escherichia coli and Shigella, but its role in Salmonella remains unknown. Our preliminary studies have revealed that speG widely affects the transcriptomes of infected in vitro M and Caco-2 cells and that it is required for the intracellular replication of Salmonella enterica serovar Typhimurium (S. Typhimurium) in HeLa cells. In this study, we demonstrated that speG plays a time-dependent and cell type-independent role in the intracellular replication of S. Typhimurium. Moreover, high-performance liquid chromatography (HPLC) of four major polyamines demonstrated putrescine, spermine, and cadaverine as the leading polyamines in S. Typhimurium. The deletion of speG significantly increased the levels of the three polyamines in intracellular S. Typhimurium, suggesting the inhibitory effect of speG on the biosynthesis of these polyamines. The deletion of speG was associated with elevated levels of these polyamines in the attenuated intracellular replication of S. Typhimurium in host cells. This result was subsequently validated by the dose-dependent suppression of intracellular proliferation after the addition of the polyamines. Furthermore, our RNA transcriptome analysis of S. Typhimurium SL1344 and its speG mutant outside and inside Caco-2 cells revealed that speG regulates the genes associated with flagellar biosynthesis, fimbrial expression, and functions of types III and I secretion systems. speG also affects the expression of genes that have been rarely reported to correlate with polyamine metabolism in Salmonella, including those associated with the periplasmic nitrate reductase system, glucarate metabolism, the phosphotransferase system, cytochromes, and the succinate reductase complex in S. Typhimurium in the mid-log growth phase, as well as those in the ilv-leu and histidine biosynthesis operons of intracellular S. Typhimurium after invasion in Caco-2 cells. In the present study, we characterized the phenotypes and transcriptome effects of speG in S. Typhimurium and reviewed the relevant literature to facilitate a more comprehensive understanding of the potential role of speG in the polyamine metabolism and virulence regulation of Salmonella.

Keywords: RNA microarray; Salmonella Typhimurium; flagella; intracellular replication; motility; polyamine; speG; transcriptome.

Figure 1

Intracellular bacterial replication assays yielded the intracellular bacterial concentrations of S. Typhimurium SL1344 and its ΔspeG mutant at various time points after invasion in different human cell lines. Human cell lines were infected with S. Typhimurium wild-type SL1344, its ΔspeG mutant, and the speG-complemented strain ΔspeG′ (multiplicity of infection = 5) for 1 h. The extracellular bacteria were killed using gentamicin after another 2 h, and the intracellular bacteria in the infected cells were allowed to proliferate for additional 7 h (output pool B1), 10 h (output pool B2), or 15 h (output pool B). (A) S. Typhimurium ΔspeG was non-significantly attenuated in output pools B1 and B2, in which intracellular bacteria in HeLa cells had been maintained for 10 and 13 h, respectively. (B–E) S. Typhimurium ΔspeG was significantly attenuated in output pool B, in which intracellular bacteria had been maintained in HeLa, Caco-2, LS174T, and THP-1 cells for 18 h, as indicated by asterisks (^*p < 0.05; n = 3).

Figure 2

HPLC quantification of cellular polyamines in S. Typhimurium SL1344 and ΔspeG before and after invasion in Caco-2 cells for 18 h. By using the same protocol as that for obtaining output pool B in the intracellular bacterial replication assay, the cellular polyamines of S. Typhimurium SL1344 and ΔspeG before (A,B) and after (C,D) 18-h intracellular internalization in Caco-2 cells were extracted through TCA precipitation and quantified through HPLC for measuring the four major polyamines. The concentration of each polyamine in S. Typhimurium was compared between the SL1344 and ΔspeG strains extracellularly and intracellularly (E–H). For the same strain of SL1344 or ΔspeG, the concentration of each polyamine was also compared between extracellular bacteria from mid-log cultures and intracellular bacteria after invasion in Caco-2 cells. Statistical significances in the comparisons are indicated by asterisks (^*p < 0.05, ^**p < 0.01, and ^***p < 0.001; n = 3).

Figure 3

Polyamine suppression assays of putrescine, cadaverine, and spermine in intracellular replication of S. Typhimurium SL1344 in Caco-2 cells. By using the same protocol as that for obtaining output pool B of the intracellular bacterial replication assay, confluent Caco-2 cells in 12-well plates were infected with overnight cultures of S. Typhimurium SL1344, and the infected cells were treated with putrescine, spermine, and cadaverine during the last 15 h in concentrations estimated from the HPLC quantification analysis. Statistical significances in the intracellular bacterial concentrations of S. Typhimurium SL1344 between the treated and untreated groups are indicated by asterisks (^*p < 0.05 and ^**p < 0.01; n = 3).

Figure 4

Quantitative real-time polymerase chain reaction analysis of 17 selected genes for validation of the RNA microarray data. (A) The mRNA expression levels of the six flagellar genes and four fimbrial genes were significantly downregulated in S. Typhimurium ΔspeG relative to its parental wild-type strain SL1344. (B) The mRNA expressions of ilvC, leuD, hisG, and SL1344_2430 were significantly upregulated and those of smvA, pyrE, and speG were significantly downregulated in intracellular S. Typhimurium ΔspeG compared with S. Typhimurium SL1344 after invasion in Caco-2 cells for 18 h. Statistical significances in mRNA expression of the selected genes between S. Typhimurium SL1344 and ΔspeG are indicated by asterisks (^*p < 0.05, ^**p < 0.01, ^***p < 0.001; n = 3).

Figure 5

Transmission electron micrographs of S. Typhimurium SL1344 and ΔspeG. Transmission electron micrographs after negative staining revealed the morphology of (A,B) S. Typhimurium SL1344 and (C,D) ΔspeG. Numerous long flagella were observed in S. Typhimurium SL1344 [magnification: (A) 10,000× and (B) 22,500×]. Only a small number of fragmented flagella were observed in S. Typhimurium ΔspeG [magnification: (C) 10,000× and (D) 22,500×].

Figure 6

Bacterial motility assays of S. Typhimurium SL1344 and ΔspeG. The motilities of S. Typhimurium SL1344 (A), ΔspeG (B), ΔspeG′ (C), ΔspaS (D), and ΔfliC (E) were examined by bacterial inoculation on semisolid agar plates with 6-h incubation at 37°C. S. Typhimurium SL1344, ΔspeG′, and ΔspaS similarly exhibited the maximal diameters of their motility zones. The motility zone of ΔspeG was smaller than that of the aforementioned strains, but still larger than that of ΔfliC.

Figure 7

Polyamine-suppressing bacterial motility assays of S. Typhimurium SL1344. The motilities of S. Typhimurium SL1344 were examined by bacterial inoculation on semisolid agar plates supplemented with four polyamines after 6-h incubation at 37°C. The motility zones of S. Typhimurium SL1344 in the semisolid LB agar plates supplemented with putrescine (625 μM; C), cadaverine (125 μM; E), and spermidine (6 μM; G) were smaller than that of S. Typhimurium SL1344 in the plates containing no polyamine (A). The motility zones of S. Typhimurium SL1344 were slightly inhibited by cadaverine (62.5 μM; D), but not suppressed by putrescine (312.5 μM; B), spermidine (3 μM; F), and two concentrations of spermine (187.5 μM; H and 375 μM; I) in the semisolid LB agar plates.