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Host-pathogen interactions : the impact of Nrampl on Salmonella enterica serovar tyuphimurium virulence gene expression Zaharik, Michelle Louise

Abstract

Nrampl (natural resistance-associated macrophage protein 1) 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 Nrampl and pathogens during infection. To explore these interactions we examined the effect of Nrampl on selected S. Typhimurium (STM) genes. Nrampl is believed to be a divalent cation transport system responsible for transport of Fe2+ and Mn2+from the phagolysosome to the cytoplasm of macrophages. We chose to investigate the role of the bacterial Mn2+ transporters MntH and SitABCD in STM pathogenesis. Nrampl-transfected cell lines infected with STM harboring mntH and sitA transcriptional fusions were used to investigate the intracellular patterns of expression of these STM genes. Both genes are expressed in the intracellular environment and are further upregulated in the presence of Nrampl. Analyses using axenic bacterial cultures demonstrated that expression of mntH and sitA responds to levels of Fe2+ and Mn2+ through the regulators Fur and MntR, while expression of mntH also responds to hydrogen peroxide through the regulator OxyR. Studies using strains deleted for oxyR or an expression plasmid deleted for the OxyR box indicated that hydrogen peroxide is important for initial expression of mntH inside macrophages both in the presence and absence of Nrampl. Similar studies investigating the role of Fur and MntR on mntH expression in cultured cells were inconclusive. Virulence studies using congenic Nrampl knockout mice demonstrated that both mntH and sitA are essential for virulence of STM in Nrampl+/+ animals. An mntH sitA double knockout strain was more attenuated than either single knockout strain, indicating that mntH and sitA contribute independently to the pathogenic nature of STM. The effect of Nrampl was also studied on selected STM virulence genes associated with the Type III Secretion System encoded within Salmonella pathogenicity island-2 (SPI2). In vivo studies demonstrated that SPI2 is essential for the ability of STM to replicate in the spleen of Nrampl+/+ mice. To investigate a potential interaction between SPI2 and Nrampl, Nrampl-transfected cell lines were used to identify that the SPI2-associated genes ssrA, sseA and sseJ, but not phoP or the SPI1 regulator hilA, are upregulated in the presence of Nrampl. Studies using axenic cultures indicate that these SPI2-associated genes are responding to extracellular levels of Fe2+ via a novel iron-responsive regulatory system. Further, these genes are differentially regulated by the global regulatory systems PhoPQ and OmpR, suggesting a previously uncharacterized complexity of SPI2 gene regulation. Overall STM is directly influenced on the genetic level by the presence of Nrampl. This impact on STM virulence gene expression correlates with the proposed function of Nrampl as a divalent cation transport system. We propose that upregulation of STM virulence genes in response to limiting levels of divalent cations is a pathogenic strategy developed by the bacterium to maintain some level of replication in naturally resistant (i.e. Nrampl+/+) hosts. We discuss how this may be involved in the development of chronic disease states by Salmonellae.

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