| Summary: | <i>Salmonella enterica</i> expresses two virulence-related type III secretion systems (T3SSs) encoded in <i>Salmonella</i> pathogenicity island 1 (SPI1) and SPI2, respectively. SseK1 is a poorly characterized substrate of the SPI2-encoded T3SS. Here, we show that this effector is essential to get full virulence both in oral and intraperitoneal mice infections, in spite of not having a role in invasion or intracellular proliferation in cultured mammalian cells. <i>In vitro</i>, expression of <i>sseK1</i> was higher in media mimicking intracellular conditions, when SPI2 was induced, but it was also significant under SPI1 inducing conditions. A detailed analysis of translocation of SseK1 into host cells unveiled that it was a substrate of both, T3SS1 and T3SS2, although with different patterns and kinetics depending on the specific host cell type (epithelial, macrophages or fibroblasts). The regulation of the expression of <i>sseK1</i> was examined using <i>lacZ</i> and bioluminescent <i>lux</i> fusions. The two-component system PhoQ/PhoP is a positive regulator of this gene. A combination of sequence analysis, directed mutagenesis and electrophoretic mobility shift assays showed that phosphorylated PhoP binds directly to the promoter region of <i>sseK1</i> and revealed a PhoP binding site located upstream of the predicted -35 hexamer of this promoter.
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