Proteomics Profiling Reveals Regulation of Immune Response to Salmonella enterica Serovar Typhimurium Infection in Mice

Abstract

Regulation of the immune response to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection is a complex process, influenced by the interaction between genetic and environmental factors. Different inbred strains of mice exhibit distinct levels of resistance to S. Typhimurium infection, ranging from susceptible (e.g., C57BL/6J) to resistant (e.g., DBA/2J) strains. However, the underlying molecular mechanisms contributing to the host response remain elusive. In this study, we present a comprehensive proteomics profiling of spleen tissue from C57BL/6J and DBA/2J strains with different doses of S. Typhimurium infection by tandem mass tag labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (TMT-LC/LC-MS/MS). We identified and quantified 3,986 proteins, resulting in 475 differentially expressed proteins (DEPs) between C57BL/6J and DBA/2J strains. Functional enrichment analysis unveiled that the mechanisms of innate immune responses to S. Typhimurium infection could be associated with several signaling pathways, including the interferon (IFN) signaling pathway. We experimentally validated the roles of the IFN signaling pathway in the innate immune response to S. Typhimurium infection using an IFN-γ neutralization assay. We further illustrated the importance of macrophage and proinflammatory cytokines in the mechanisms underlying the resistance to S. Typhimurium using quantitative reverse transcription-PCR (qRT-PCR). Taken together, our results provided new insights into the genetic regulation of the immune response to S. Typhimurium infection in mice and might lead to the discovery of potential protein targets for controlling salmonellosis.

Keywords: S. Typhimurium; Salmonella; genetic regulation; immune response; mass spectrometry; mouse; proteome; proteomics.

FIG 1

Proteomic profiling of spleens from C57BL/6J (B6) and DBA/2J (D2) mice after Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. (A) Schematic diagram of proteomics experiments. B6 and D2 mice were infected with S. Typhimurium strain 14028GFP by oral administration at two dosages (5 × 10⁶ and 5 × 10⁸ CFU) for 14 days for individual males and females of each mouse strain. Whole-proteome analysis was performed by two-dimensional liquid chromatography-tandem mass spectrometry (TMT-LC/LC-MS/MS). (B) Principal-component analysis (PCA) showing high correlation between biological replicates and clear separation between groups; (C) scatter density plot showing correlation of proteins within groups and between groups. Point density from low to high is indicated by the color gradient from yellow to violet.

FIG 2

Differential protein expression and pathway analyses. (A) Volcano plot showing 475 DEPs between C57BL/6J (B6) and DBA/2J (D2) mice after infection with high-dose S. Typhimurium; (B) heat map of differentially expressed proteins (DEPs). A total of 118 DEPs were detected from the interaction effects of strain and dosage by two-way analysis of variance (ANOVA) (<5% FDR). (C) Bubble plot showing Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by DEPs in spleen between B6 and D2 mice infected by high-dose S. Typhimurium; (D) protein-protein interaction (PPI) network in lysosome and phagosome pathways. The color of the dot represents the value of the −log₂ fold change of DEPs in spleens between B6 and D2 mice infected by high-dose S. Typhimurium. The color of the line is related to the combined score. (E) Bar plot showing expression patterns of two representative DEPs, GBP4 (guanylate-binding protein 4) and GLO1 (glyoxalase I). The two proteins displayed distinctive expression patterns among infection and control groups from the B6 and D2 strains.

FIG 3

Weighted protein coexpression network and functional enrichment analysis of proteins in coexpressed modules. (A) Workflow of coexpression analysis followed by protein-protein interaction (PPI) network modularization. Three coexpression clusters and 16 PPI modules were detected. (B) Three coexpression protein clusters were detected from 475 DEPs that showed strain difference between B6 and D2 mice infected by high-dose S. Typhimurium. (C) Heat map showing the Gene Ontology (GO) pathways significantly enriched in one of the three coexpression clusters. Enrichment analysis was performed using the R package clusterProfiler (version 3.18.1). The gradient presents the corresponding logarithmic enrichment P value. (D) Heat map showing six proteins involved in lysosome and autophagy pathways. These six proteins were significantly upregulated in D2 compared to B6 mice.

FIG 4

Functional modules identified by superimposing coexpression clusters on the protein-protein interaction (PPI) network. (A) PPI network showing interactions of 13 proteins of the interferon (IFN) signaling pathway. The PPI network was generated using the STRING database and visualized in Cytoscape. (B) Heat map showing eight proteins involved in IFN signaling pathway. The expression levels of these eight proteins showed downregulation in B6 after high-dose S. Typhimurium infection compared to the control group.

FIG 5

Gamma interferon (IFN-γ) neutralization assay to validate the role of IFN-γ in innate immune response to S. Typhimurium infection revealed by proteomics results. (A) Experimental design of IFN-γ neutralization assay; (B) Western blot analysis to validate the change of expression levels identified by the proteomics study. The B6 and D2 mice were inoculated with S. Typhimurium by oral gavage. For the IFN-γ neutralization assay, mice were intraperitoneally injected with neutralizing anti-IFN-γ antibody and the matched isotype control. Spleens were harvested from mice and homogenized by RIPA lysis with protease inhibitor cocktail. The protein concentrations of tissue lysates were quantified by BCA assay. Cell extracts were resolved on 10% SDS-PAGE and transferred to PVDF membrane. Five primary antibodies were used to probe the target proteins: STAT1, IRF3, GBP1, ISG20, and GAPDH. The membrane was then probed by goat anti-rabbit HRP-linked secondary antibody and developed with SuperSignal West Femto maximum sensitivity substrate. All four target proteins were upregulated in both B6 and D2 mice after S. Typhimurium infection compared with the corresponding controls, but the levels of upregulation in D2 mice appeared to be more pronounced than those in the B6 mice. The upregulation of these proteins in response to S. Typhimurium infection was completely abolished by anti-IFN-γ neutralizing antibody, while the upregulation could not be abrogated by the matching isotype control. (C) Representative photomicrographs of hematoxylin and eosin (H&E)-stained spleen and liver sections (magnification, ×40; bar, 250 μm). The spleens of B6 and D2 control mice had distinct and intact areas of white pulp and red pulp. The spleen of S. Typhimurium-infected B6 mice showed elevated inflammation, disrupted splenic architecture, and coalescing regions of necrosis. In contrast, the spleens of D2 mice presented minimal signs of inflammation, retained the architecture of red and white pulp, and displayed only mild multifocal necrosis. In the groups of B6 and D2 mice injected with IFN-γ neutralization antibody, the IFN-γ signaling pathway was blocked, and the spleens of B6 and D2 displayed equally severe signs of infection. The liver sections demonstrated similar patterns. Compared with the healthy liver sections from corresponding control mice, the livers of S. Typhimurium-infected B6 mice had much more inflammatory nodules and hypercellularity (extramedullary hematopoiesis) than D2 mice. However, in the IFN-γ neutralization groups, the livers of both B6 and D2 mice displayed the same extent of disruptions of histological structure and lesions.

FIG 6

Experimental validation of macrophages and proinflammatory cytokines after infection by macrophage phagocytosis assay and qRT-PCR. (A) Macrophage phagocytosis assay revealing better clearance of pathogens by macrophages from D2 mice than those from B6 ones. Macrophages were differentiated from the bone marrow cells. Fluorescent images were obtained with a Leica DMi8 Thunder fluorescence microscope (Leica Camera, Wetzlar, Germany) at 550-nm excitation for green fluorescent protein (GFP) expressed in S. Typhimurium strain 14028GFP and 395-nm excitation for DAPI. (B) qRT-PCR analysis for expression of five proinflammatory cytokines (IFN-γ, iNOS, IFN-β, IL-6, and IL-8) after S. Typhimurium infection between B6 and D2 strains by qRT-PCR (n = 2 for each group). Error bars represent mean ± standard deviation (SD). *, P < 0.05; **, P < 0.01.