Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells

Abstract

Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Y.enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (including Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (including Rab1 and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions.

Statement of significance of the study: We describe a proteome of Yersinia enterocolitica-infected HeLa cells, including a description of specific proteins differentially expressed upon infection, molecular functions as well as pathways altered during infection. This proteomic study can lead to a better understanding of Y. enterocolitica pathogenesis in human epithelial cells.

Keywords: Integrin signaling; Label-free proteomics; Pathway modeling; Protein ubiquitination; Yersinia enterocolitica infection.

Figure 1. Comparison of proteomics results obtained from HeLa cells lysed by using two different buffers

(A). The Venn diagrams illustrate the number of proteins identified in at least two of the analyzed biological replicates, and unique spectra detected in each sample type. (B). GO term analysis of subcellular localizations of unique proteins. Proteins identified in each sample type were subjected to the GO term analysis by using The Database for Annotation, Visualization and Integrated Discovery (DAVID).

Figure 2. Differential protein expression in HeLa cells infected or not infected with Yersinia enterocolitica

(A). HeLa cells were infected with Y. enterocolitica and analyzed by label-free quantitative proteomics. Significantly regulated proteins are listed, along with their fold change (infected/control), identification number, symbol, Entrez gene name, localization (suggested by Ingenuity Pathway Analysis software) and protein function. (B)–(C) HeLa cells were infected with Y. enterocolitica wild-type (B) and Caco-2 cells were infected with wild-type or plasmid-cured mutant (C) for 4 hours. The extracted proteins were analyzed by SDS-PAGE and western blotting. Anti-beta-actin antibody was used as a loading control.

Figure 3. Network and pathway analysis of differentially regulated proteins in HeLa cells subjected to Yersinia infection

(A)–(B). Differentially expressed proteins with protein level affected by Yersinia infection (Fig. 2A) were analyzed by Ingenuity Pathway Analysis to identify canonical pathways (A) and molecular functions (B). (C). The top protein network identified from differentially regulated proteins in infected cells is associated with the integrin signaling and Signaling by Rho family of GTPases. Expression of proteins is indicated by colors (red for downregulated and green for upregulated proteins, white for proteins that were not on a differentially expressed protein list). Different shades of color represent the level of regulation.

Figure 4. Protein network analysis of differentially regulated proteins in HeLa cells subjected to Yersinia infection

The second top network identified from differentially regulated proteins in Yersinia-infected HeLa cells is associated with the protein ubiquitination pathway. Expression of proteins is indicated by colors (red for downregulated and green for upregulated proteins, white for proteins that were not on a differentially expressed protein list). Different shades of color represent the level of regulation.