Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Stimulated by Mycobacterium tuberculosis PPE57 Identifies Characteristic Genes Associated With Type I Interferon Signaling

Abstract

Proline-glutamic acid (PE)- and proline-proline-glutamic acid (PPE)-containing proteins are exclusive to Mycobacterium tuberculosis (MTB), the leading cause of tuberculosis (TB). In this study, we performed global transcriptome sequencing (RNA-Seq) on PPE57-stimulated peripheral blood mononuclear cells (PBMCs) and control samples to quantitatively measure the expression level of key transcripts of interest. A total of 1367 differentially expressed genes (DEGs) were observed in response to a 6 h exposure to PPE57, with 685 being up-regulated and 682 down-regulated. Immune-related gene functions and pathways associated with these genes were evaluated, revealing that the type I IFN signaling pathway was the most significantly enriched pathway in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1. These PPE57-related transcriptomic profiles have implications for a better understanding of host global immune mechanisms underlying MTB infection outcomes. However, more studies regarding these DEGs and type I IFN signaling in this infectious context are necessary to more fully clarify the underlying mechanisms that arise in response to PPE57 during MTB infection.

Keywords: Mycobacterium tuberculosis; PPE57; RNA sequencing; peripheral blood mononuclear cell; type I interferon signaling.

Figure 1

Differentially expressed genes (DEGs) identified in PBMCs stimulated with PPE57 relative to control cells. (A) Principal component analysis of DEG transcripts. (B) A volcano plot highlighting DEGs in comparisons of control and PPE57-stimulated samples (log₁₀p-value vs log₂FC). Green and red dots with negative and positive change values respectively correspond to downregulated and upregulated DEGs. (C) Hierarchical clustering of transcripts that were significantly (≥2-fold change) upregulated (red) or downregulated (blue) in PPE57-treated PBMCs, with three repeats per sample.

Figure 2

Gene set differences between PPE57-stimulated PBMCs and controls as illustrated through a gene set enrichment analysis (GSEA) approach. Enrichment plots for six GSEA pathways that were enriched in PPE57-stimulated PBMCs relative to controls. A gene set was considered to be significantly enriched at a P ≤ 0.05.

Figure 3

GO annotations and KEGG pathway analysis results for upregulated DEGs. (A) The top 30 biological process, cellular component, and molecular function GO terms are shown (P < 0.05; unique gene number of GO terms > 2). (B) The top 20 KEGG pathways with positive enrichment are shown in a bubble chart with enriched pathways on the y-axis and enrichment scores on the x-axis. A positive correlation between bubble size and the number of pathway-related genes was observed, with a larger pathway enrichment P-value being associated with an increase in the red coloration of that bubble.

Figure 4

PPI networks and modules incorporating the top 100 DEGs from PPE57-stimulated PBMCs. (A) PPI network. (B) Module. The DEGs associated with the type I IFN signaling pathway are marked with red circles. DEGs associated with the RIG-I-like receptor signaling pathway are marked with blue circles.

Figure 5

Venn diagram demonstrating the number of common overlapping genes in GSEA, GO, and PPI analyses. These overlapping genes were related to the type I IFN signaling pathway. Genes analyzed via GSEA, GO, and PPI approaches are denoted in blue, green, and red circles, respectively. In total, 14 genes overlapped across all three of these analyses.

Figure 6

Validation of transcriptomic sequencing results by real-time qPCR.