Characterization of MgtC, a virulence factor of Salmonella enterica Serovar Typhi

Abstract

The MgtC is a virulence factor in Salmonella Typhimurium that is required for growth at low-Mg2+ concentrations and intramacrophage survival. This gene is codified in a conserved region of the Salmonella pathogenicity island 3 (SPI-3), and is also present in the chromosome of other Salmonella serovars. In this study we characterized the MgtC factor in S. Typhi, a human specific pathogen, by using mgtC and SPI-3 mutant strains. We found that MgtC is the most important factor codified in the SPI-3 of S. Typhi for growth in low-Mg2+ media and survival within human cells. In addition, by using reporter genes we determined that the low-Mg2+ concentration, acidic media and PhoP regulator induce mgtC expression in S. Typhi. We suggest that MgtC is the most important virulence factor codified in the SPI-3 of S. Typhi.

Figure 1. MgtC is necessary for growth at a low-Mg²⁺ concentration.

Strains WT (STH2370 wild type), ΔmgtC (mgtC⁻), ΔmgtC/pBmgtC (mgtC ⁺) and ΔmgtC/pBBR-5 (mgtC⁻) were grown in M9 minimal medium supplemented with 10 mM (A) or 10 µM (B) MgCl₂. The OD₆₀₀ was measured at the indicated times. Values represent the mean of three independent experiments ±SD (*p<0.05).

Figure 2. MgtC has an important role in the growth of S. Typhi within human cells.

Infection assays in U937 (A) and HEp-2 (B) human cells using WT (STH2370 wild type), ΔmgtC (mgtC⁻), ΔmgtC/pBmgtC (mgtC ⁺) and ΔmgtC/pBBR-5 (mgtC⁻) strains. Culture cells were infected at a MOI of 50∶1 (U937) and 100∶1 (HEp-2), respectively. Colonies were counted at time 2 h and 24 h and expressed as a percentage of intracellular survival. Values represent the mean of at least three independent experiments ±SD (*p<005).

Figure 3. MgtC can restore the WT phenotype in a SPI-3 mutant strain.

Strains WT (STH2370 wild type), ΔSPI-3 (SPI-3⁻), ΔSPI-3/pBmgtC (mgtC ⁺) and ΔSPI-3/pBBR-5 (SPI-3⁻). (A) For growth in 10 µM MgCl₂ strains were incubated in M9 minimal medium and the OD₆₀₀ was measured at the indicated times. (B) U937 cells were infected at a MOI of 50∶1. Colonies were counted at time 2 h and 24 h and expressed as a percentage of intracellular survival. Values represent the mean of at least three independent experiments ±SD (*p<0.05).

Figure 4. The PhoP regulator controls the expression of mgtC in S. Typhi.

The strains used were WT (STH2370 wild type), ΔphoP (phoP::cam), ΔphoP/pBphoPQ (phoPQ ⁺) and ΔphoP/pBBR-5 (phoPQ ⁻). The cultures were grown in acidic LB medium (pH 5) for 5 h, and the samples taken had an OD₆₀₀ ranging from 0.3 to 0.6. The expression of mgtC was evaluated using three methods: A, β-Galactosidase assay with strains carrying the reporter lacZ gene downstream of the mgtC promoter. Values represent the mean of at least three independent experiments ±SD (*p<0.05). B, RT-PCR assay. C, Immunoblot method using a STH2370 MgtC-3×Flag epitope-tagged strain grown on LB broth at both pH 5 and pH 7. D, Immunoblot method using strains carrying the MgtC-3×Flag epitope tag and grown in acidic LB medium (pH 5) for 5 h.