PNNL

Abstract

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium (STM) must replicate within host macrophages in order to establish systemic infection in susceptible mice. In an effort to identify new STM proteins that help the bacterium colonize macrophages, we have cultured STM cells with a low pH/low magnesium medium (MgM) under two different conditions termed MgM-Shock and MgM-Dilution and investigated the impacts of these culturing conditions on the STM proteome by using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. LC-MS/MS results showed that alteration of culturing conditions affected a group of STM proteins differently. Compared to MgM-Shock, MgM-Dilution induced more proteins of the Salmonella-pathogenecity island 2-type III secretion system (SPI2-T3SS). The abundances of the proteins used for cobalamin biosynthesis increased under MgM-Shock condition but decreased under MgM-Dilution condition, while those proteins used for thiamine or biotin biosynthesis were not affected under the former condition but increased under the latter condition. Western-blot (WB) analysis confirmed the LC-MS/MS results. Because cobalamin, thiamine and biotin play different roles in STM metabolism, differential induction of the proteins involved in their biosyntheses suggests that the metabolic states of STM cells under these conditions differ considerably. WB analysis also showed that the abundances of SPI2-T3SS proteins SsaQ and SseE and biotin biosynthesis proteins BioB and BioD increased after STM infection of RAW 264.7 macrophages. Deletion of the gene encoding BioB reduced the ability of STM to replicate inside the macrophages, demonstrating for the first time the involvement of a biotin synthesis protein in STM colonization of macrophages.