Salmonella pathogenesis is dependent on its ability to invade and replicate within a variety of host cells. Upon bacterial uptake by macrophages, maturation of the Salmonella Containing Vacuole (the SCV) initiates. The process of SCV maturation depends on the interactions between Salmonella effectors and host cell regulators and is a prokaryotic developmental program that follows a strict temporal and spatial path. Immediately following invasion, individual Salmonella cells are found within discrete vacuoles. Subsequently, intracellular bacterial replication begins after an initial lag period and is accompanied by the formation of extensive membrane tubules (Salmonella-induced filaments, Sifs), which project from the SCVs and extend throughout the host cell. These events are coupled to interactions with host cell components and can be divided into discrete temporal and spatial steps: (1) Initial contact with the host cell, (2) Formation of early SCV, (3) Maturation into late SCV, (4) Initiation of bacterial replication, (5) Formation of Sifs.

In this project, we have constructed a set of reporter fusions which are temporally and spatially regulated during the progression of SCV maturation. The reporter fusions were constructed using Red-mediated recombination (1) and the promoters were selected from the recently published expressional data of Salmonella infection of murine macrophages (2). As reporter proteins we both use a stable GFPmut3 variant as well as an unstable GFP variant (3). Using these fusions in combination with available host markers it will be possible to estimate the time-point at which a specific gene is required for progression of SCV maturation. These developmentally regulated reporter fusions constitute a set of novel developmental markers for the study of Salmonella Typhimurium infection of murine macrophages.

Reference List

   1.   R. G. Gerlach, S. U. Holzer, D. Jackel, M. Hensel, Appl. Environ. Microbiol. 73, 4234 (2007).

   2.   S. Eriksson, S. Lucchini, A. Thompson, M. Rhen, J. C. Hinton, Mol. Microbiol. 47, 103 (2003).

   3.   J. B. Andersen et al., Appl. Environ. Microbiol. 64, 2240 (1998).