Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium

Abstract

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food--and water--borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

Figure 1. Glycolysis is important but not essential for the invasion and intracellular replication of HeLa cells with S. Typhimurium.

(A) Invasion assay of S. Typhimurium 4/74 parental and ΔpfkAB (JH3486) strains in HeLa cells (B). Intracellular replication assays of S. Typhimurium 4/74 parental and ΔpfkAB (JH3486) strains during infection of HeLa cells. The chart shows the percentage replication of bacteria between 2 h and 6 h. (C) Complementation of invasion of the S. Typhimurium ΔpfkAB strain in HeLa cells. (D) Complementation of intracellular replication of the S. Typhimurium ΔpfkAB strain in HeLa cells. Each bar represents the statistical mean from three biological replicates and the error bars represent the standard deviation. (The significant differences between the parental 4/74 strain (A, B), or the 4/74 (pWKS30) strain (C, D) and the mutant strains are shown by asterisks, *p<0.05. **p<0.01, and ***p<0.001).

Figure 2. Summary of glucose transport in Salmonella .

Glucose can be taken up by the EII^Glc and/or the EII^Man transporters and simultaneously phosphorylated to generate glucose-6-phosphate. The EII^Glc PTS transporter is encoded by two genes; crr encodes the IIA^Glc protein whilst ptsG encodes the membrane-bound IIBC^Glc protein. The EII^Man PTS transporter is encoded by three genes, manX, manY and manZ that encode the IIAB^Man, and the IIC^Man and IID^Man components of the transporter system, respectively. In order to transport and phosphorylate glucose, the EII^Glc and EII^Man transporters require phosphate donated from phosphoenol-pyruvate (PEP) via the EI and HPr phospho-relay proteins that are encoded by ptsI and ptsH, respectively. In addition, unphosphorylated glucose can be imported by the GalP and/or MglABC transporters then subsequently phosphorylated by glucose kinase (Glk) to produce glucose-6-phosphate.

Figure 3. Hierarchical clustering of 53 S. Typhimurium PTS genes expressed during infection of HeLa cells.

The filtered data was clustered according to similarity of expression level using the standard correlation tool in GeneSpring GX7.3^™ (Agilent). Each gene is colour-coded according to the level of expression (i.e. signal ratio of cDNA versus genomic DNA). Highly expressed genes are shown in red and weakly expressed genes are dark blue. The clustering map was compiled from microarray data deposited at ArrayExpress (accession number E-MEXP-1368) and described in .

Figure 4. The PTS-system is important but not essential for the invasion and intracellular replication of HeLa cells in S. Typhimurium.

Invasion (A) and intracellular replication (B) of S. Typhimurium 4/74, ΔptsHI (JH3537), ΔptsHIcrr (JH3536) and Δcrr (JH3502), strains during infection of HeLa cells. (A) The chart shows the numbers of viable bacteria (expressed as percentages of the initial inoculum) within host cells at 2 h post-infection. (B) The chart shows the percentage replication of bacteria between 2 h and 6 h. Each bar represent the statistical mean from three biological replicates and the error bars represent the standard deviation (The significant differences between the parental 4/74 strain and the mutant strains are shown by asterisks *p<0.05, **p<0.01, and ***p<0.001).

Figure 5. Glucose transport is required for efficient intracellular replication of S.Typhimurium in HeLa cells.

Invasion (A) and intracellular replication (B) of S. Typhimurium 4/74, ΔptsG (JH3504), ΔmanXYZ (JH3501), Δglk (JH3494), ΔptsGΔmanXYZ (AT1011), ΔptsG Δglk (AT1012), ΔmanXYZ Δglk (AT1013), and ΔptsGΔmanXYZΔglk (AT1014) strains during infection of HeLa cells. (A) The chart shows the numbers of viable bacteria (expressed as percentages of the initial inoculum) within host cells at 2 h after infection. (B) The chart shows the percentage replication of bacteria between 2 h and 6 h. Each bar indicates the statistical mean for three biological replicates, and the error bars indicate the standard deviations. The significant differences between the parental 4/74 strain and the mutant strains are shown by asterisks *p<0.05, **p<0.01, and ***p<0.001.

Figure 6. S. Typhimurium does not require the glyoxylate shunt or gluconeogenesis for intracellular replication within HeLa cells.

Invasion (A) and intracellular replication (B) of S. Typhimurium 4/74, ΔaceA (JH3385), and ΔppsΔpckA (JH3487) strains during infection of HeLa cells. (A) The chart shows the numbers of viable bacteria (expressed as percentages of the initial inoculum) within host cells at 2 h after infection. (B) The chart shows the percentage replication of bacteria between 2 h and 6 h. Each bar represents the statistical mean from two biological replicates (performed in triplicate) and the error bars represent the standard deviation.