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.
Highlights
Brucella spp are facultative intracellular, Gram-negative coccobacilli belonging to the alpha subclass of Proteobacteria
Analysis of digital gene expression (DGE) libraries We investigated molecular changes taking place in mouse peritoneal macrophages following infection with virulent and vaccine strains of Brucella
Global gene expression profiles were analyzed using the Solexa/Illumina DGE system, a tag-based transcriptome sequencing method. cDNA libraries from uninfected macrophages (Mc) and from macrophages infected with strains M28 or M5-90 were sequenced using massively parallel sequencing on the Illumina platform
Summary
Brucella spp are facultative intracellular, Gram-negative coccobacilli belonging to the alpha subclass of Proteobacteria They reside in professional and non-professional phagocytes, and macrophages in particular are a major target in infected mammals. The attenuated B. melitensis vaccine smooth strain M5-90 was derived from the virulent smooth B. melitensis M28 ovine isolate by serial passage in chicken, acriflavine treatment, and further passage for 90 generations in chicken embryo fibroblasts [5]. This attenuated live vaccine is considered to be one of the key factors that caused a rapid decline in the incidence of brucellosis in animals and humans in China. The underlying molecular or physiological changes underlying loss of virulence are not understood
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