Deep-sequencing analysis of the mouse transcriptome response to infection with Brucella melitensis strains of differing virulence

Abstract

Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE) analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK) pathways. Further analysis employed gene ontology (GO) analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy.

Figure 1. Comparative analysis of tag-mapped transcripts in the three libraries.

A Venn diagram was created to quantify the overlap in the response. 10,171 transcripts were identified in both libraries, and 392 and 516 transcripts were present in the M28 and M5-90 libraries, respectively.

Figure 2. Differentially expressed genes between libraries.

All genes mapped to the genome were examined for expression differences between the different libraries. Numbers of differentially expressed genes represent across sense transcripts, using threshold values FDR≤0.001 and log2 Ratio≥1 for controlling false discovery rates.

Figure 3. DGE (M28 versus M5-90 libraries) GO enrichment chart.

The upper chart comprises GOs targeted by overexpressed DEGs; the lower chart comprises GOs targeted by downregulated DEGs. All these GOs show increased enrichment. The vertical axis is the GO category and the horizontal axis is the enrichment of GO.

Figure 4. Path-Net of the significant pathways in the M28 versus M5-90 libraries (P<0.05).

18 out of the 29 significant pathways were chosen to build the Path-Net; potential interactions between the pathways were evaluated using Degree. Red dots represent significant pathways, grey dots represent other relevant pathways. The lines represent the interaction of the pathways.

Figure 5. Signal-Net of the DEGs of the significant pathways of the M28 versus M5-90 libraries (P<0.05).

Red and grey dots represent up- and downregulated genes, respectively; area sizes represent Degree, and lines represent interactions between the gene products.

Figure 6. qPCR validation of the DGE data.

Relative qPCR was used to measure changes in target gene expression in infected macrophages relative to reference samples. Results are expressed as the target/reference ratio of each sample normalized to the reference gene Fnip1. (A–C). Genes not detected on the 2D-DIGE. Error bars, standard error; TPM, transcripts per million mapped reads.