Cellular Microbiology (2001) 3(9), 587±597 Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo Suzana P. Salcedo, 1 Mahdad Noursadeghi, 2 Jonathan Cohen 2 and David W. Holden 1 * 1 Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College School of Medicine, The Flowers Building, Armstrong Road, London SW7 2AZ, UK. 2 Department of Infectious Diseases, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. Summary We used flow cytometry and confocal immunofluor- escence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraper- itoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA 2 mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intra- cellular replication in vivo. Introduction Salmonella typhimurium causes a systemic typhoid-like illness in mice. In susceptible (Ity s ) host strains, less than 10 wild-type bacteria administered by the subcutaneous, intravenous or intraperitoneal route are sufficient to cause a fatal infection (Plant and Glynne, 1974; Shea et al., 1999). After oral inoculation, translocation of S. typhimurium across the gut epithelium into the bloodstream occurs by invasion of M cells in ileal Peyer's patches (Carter and Collins, 1974; Jones et al., 1994), and also via transmigrat- ing CD18-expressing phagocytes (Vazquez-Torres et al., 1999). Regardless of the route of inoculation, a transient bacteraemia is followed over the course of several days by the accumulation of large numbers of bacteria within the spleen and liver, leading to a second, fatal bacteraemia (Carter and Collins, 1974; Shea et al., 1999). Despite widespread use of this murine model of systemic infection, the localization of S. typhimurium in vivo has been contentious (Wang et al., 1988; Conlan and North, 1992; Dunlap et al., 1992; Matsui et al., 2000). In confocal microscopic analysis of liver sections, bacteria were shown clearly to be present within CD18-expressing cells presumed to be macrophages (Richter-Dahlfors et al., 1997). However, data regarding the localization of Salmonella within the spleen, which is the other major site of S. typhimurium replication in vivo, remain inconclusive. Previous studies have suggested different locations, ranging from extracellular spaces to polymorphonuclear cells (Wang et al., 1988; Dunlap et al., 1992). S. typhimurium has also been shown to reside in CD11b- positive cells, which include macrophages, monocytes, polymorphonuclear cells and even some subsets of lymphocytes (Matsui et al., 2000). In vitro studies, using different host cell types, have shown that S. typhimurium replicates intracellularly within Salmonella-containing vacuoles (SCVs) apparently pro- tected from host cell antimicrobial activities (Buchmeier and Heffron, 1991; Rathman et al., 1997; Vazquez-Torres et al., 2000). Genetic studies have led to the identification of a large number of genes necessary for intracellular survival. These include the phoP/Q genes, encoding a two-component regulatory system, which regulates the expression of over 40 genes (Miller and Mekalanos, 1990), the spv locus on the large S. typhimurium plasmid (Gulig et al., 1998) and the Salmonella pathogenicity island-2 (SPI-2), which encodes a type III secretion system (TTSS) needed for bacterial replication within cultured macrophages (Ochman et al., 1996; Cirillo et al., 1998). The fact that strains carrying mutations in genes required for intracellular growth in vitro are typically avirulent in mice (Fields et al., 1986) suggests that S. typhimurium replicates intracellularly in vivo. Q 2001 Blackwell Science Ltd Received 2 March, 2001; revised 14 May, 2001; accepted 21 May, 2001. *For correspondence. E-mail d.holden@ic.ac.uk; Tel. (144) 020 7594 3073; Fax (144) 020 7594 3076.