Inorganic polyphosphate (polyP) deficiency in enteric bacterial pathogens reduces their ability to invade and establish systemic infections in different hosts. For instance, inactivation of the polyP kinase gene () encoding the enzyme responsible for polyP biosynthesis reduces invasiveness and intracellular survival of serovar Typhimurium (. Typhimurium) in epithelial cells and macrophages . In addition, the virulence of a . Typhimurium Δ mutant is significantly reduced in a murine infection model. In spite of these observations, the role played by polyP during the -host interaction is not well understood. The social amoeba has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In fact, many intracellular pathogens can survive within cells using molecular mechanisms also required to survive within macrophages. Recently, we established that . Typhimurium is able to survive intracellularly in and identified relevant genes linked to virulence that are crucial for this process. The aim of this study was to determine the effect of a polyP deficiency in . Typhimurium during its interaction with . To do this, we evaluated the intracellular survival of wild-type and Δ strains of . Typhimurium in and the ability of these strains to delay the social development of the amoeba. In contrast to the wild-type strain, the Δ mutant was unable to survive intracellularly in and enabled the social development of the amoeba. Both phenotypes were complemented using a plasmid carrying a copy of the gene. Next, we simultaneously evaluated the proteomic response of both . Typhimurium and during host-pathogen interaction via global proteomic profiling. The analysis of our results allowed the identification of novel molecular signatures that give insight into - interaction. Altogether, our results indicate that inorganic polyP is essential for . Typhimurium virulence and survival in . In addition, we have validated the use of global proteomic analyses to simultaneously evaluate the host-pathogen interaction of . Typhimurium and . Furthermore, our infection assays using these organisms can be exploited to screen for novel anti-virulence molecules targeting inorganic polyP biosynthesis.