Host-pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes

Abstract

Nramp1 (Natural resistance-associated macrophage protein-1; also known as Slc11a1) is a host resistance gene that provides protection against several intracellular pathogens, including Salmonella enterica serovar Typhimurium. Little is known about the dynamic interplay that occurs between mammalian host resistance determinants such as Nramp1 and pathogens during infection. To explore these interactions, we examined the effect of Nramp1 on expression of Salmonella typhimurium (STM) virulence factors. We demonstrate that Salmonella pathogenicity island 2 (SPI2) is essential for replication of STM in spleens of infected Nramp1(+/+) mice. Furthermore, the presence of Nramp1 in transfected cell lines and congenic knockout mice resulted in the up-regulation of STM SPI2-associated virulence genes critical for intramacrophage survival. This Nramp1-dependent up-regulation of SPI2 was mimicked in vitro by chelation of iron, demonstrating the iron-responsive nature of expression of STM SPI2-associated virulence genes. We propose that acquisition of SPI2 by S. enterica not only enabled this bacterium to become an effective intracellular pathogen but also allowed the bacterium to withstand the effects of macrophage defense mechanisms such as Nramp1 early in the evolution of its pathogenic character. These dynamic Nramp1-pathogen interactions may be essential for regulating the course of an infection. This study demonstrates the presence of a previously undescribed direct influence of a mammalian innate host resistance locus on a pathogen at the genetic level.

Fig 1.

SPI2 permits replication of STM in murine spleen in the presence of Nramp1. Age- and sex-matched congenic 129sv/J mice were infected i.p. with 1e5 cfu of either wild-type or SPI2- (ssrA:Km) STM 14028s and spleens were harvested for bacterial enumeration at designated time points. Data represent the average ± the SEM of two experiments performed on experimental groups of five mice.

Fig 2.

Presence of Nramp1 in transfected RAW264.7 cells leads to the up-regulation of SPI2 and SPI2-associated virulence genes. Cells were infected with STM14028s carrying the plasmids containing the different transcriptional fusions. Patterns of expression over a 24-h time course are shown for the SPI1 regulatory gene hilA, the pleiotropic virulence regulatory gene phoP, the locus encoding the kinase for the two-component regulatory system that regulates SPI2, ssrA, the SPI2-encoded effector locus sseA, and the SP12 effector locus sseJ in RAW264.7 cell lines transfected with empty pCB6 vector (Nramp1−) or with pCB6 carrying a constitutively expressed wild-type Nramp1 gene (Nramp1+). These data are expressed as the average ratio of expression normalized to the initial observed level of expression at 2 h postinfection in the Nramp1− cell line and represent the mean ± the SEM of five separate experiments performed in duplicate. Statistical significance (P < 0.05) was determined by using the Wilcoxon rank sum test for unpaired samples.

Fig 3.

Expression of SPI2 and SPI2-associated genes is up-regulated in spleens of STM-infected Nramp1^+/+ 129sv mice. An average of 2.5e5 STM 14028s bearing the different transcriptional fusion plasmids were injected i.p. into age- and sex-matched 129sv mice and spleens were harvested at 24 h postinfection for evaluation of β-galactosidase activity. These data represent the mean ± the SEM of five separate experiments performed on groups of five mice per fusion of interest. Statistical significance (P < 0.05) was determined using the Wilcoxon rank sum test for unpaired samples.

Fig 4.

(A) Chelation of divalent cations by various chelators results in up-regulation of STM virulence genes. Expression levels are shown for hilA, phoP, ssrA, sseA, and sseJ in N minimal media alone, or N minimal media supplemented with one of the following chelators: EGTA, EDTA, or dipyridyl. Statistical significance (+, P < 0.05; *, P < 0.01) was determined by using Student's t test for paired samples. (B) Chelation of divalent cations results in up-regulation of SPI2 and SPI2-associated genes in vitro. Expression levels in N minimal media alone, N minimal media supplemented with the chelator dipyridyl, or dipyridyl plus one of the following divalent cations MgSO₄, MnSO₄, or FeSO₄, are shown for hilA, phoP, ssrA, sseA, and sseJ. Statistical analyses reflect differences in expression levels between cultures grown in dipyridyl-chelated media versus cultures grown in media supplemented with dipyridyl and the indicated excess divalent cation. Statistical significance (+, P < 0.05; *, P < 0.01) was determined by using Student's t test for paired samples.