Proteomic Analysis of Salmonella enterica Serovar Typhimurium Isolated from RAW 264.7 Macrophages

Abstract

To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistanceassociated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118 -3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

“…Overall, our work significantly advanced quantitative proteomics of intracellular Salmonella, as only 315 bacterial proteins were measured previously (19). With ϳ3,300 proteins identified in total, our LC-MS-based approach covered ϳ73% of the Salmonella proteome, which contains ϳ4,500 proteins.…”

“…Further mutational analyses revealed that most of these observed proteins/metabolic enzymes are nonessential for Salmonella virulence, suggesting robust bacterial metabolism and availability of diverse nutrients in the host. In the same year, Shi et al reported the first quantitative time course study of intracellular Salmonella proteome in infected macrophages (19). They found that 39 of 315 bacterial proteins were strongly induced after infection and, notably, that deletion of STM3117 led to markedly reduced replication, implicating STM3117 in a critical role in Salmonella colonization inside macrophages.…”

Quantitative proteomics of intracellular Porphyromonas gingivalis

“…This was significantly higher coverage relative to other literature reports for intracellular pathogens. For example, in a recent study of Salmonella enterica serovar Typhimurium recovered from macrophages, 315 proteins were detected and 39 were described as differentially regulated [42].…”

Orthogonal Alkynyl Amino Acid Reporter for Selective Labeling of Bacterial Proteomes during Infection

“…[3] The large excess of host proteins in mixed pathogen-host lysates presents a significant challenge for proteomic analysis of bacterial proteins during infections [3] and even after physical isolation of intact bacteria significant amounts of host proteins still remain. [4,5] This is particularly important since many bacterial virulence factors are often expressed at low levels. [2] New strategies are therefore required to selectively enrich bacterial proteins from host proteomes for their analysis during infection.…”