Attenuation Molecular Basis Of Brucella Melitensis Vaccine Strain M5-90

Brucella melitensis is a Gram-negative coccobacillus bacteria belonging to theα-Proteobacteria subclass. It is an important zoonotic pathogen that causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. So far, no satisfactory vaccine has been discovered to fight the disease in humans, while animal vaccines are pathogenic to humans. Therefore, we should venture deeper in the study of the pathogenic and attenuation mechanism of Brucella as well as the development of new types of Brucella vaccines.I. molecular basis of Attenuation of Brucella melitensis strain M5-90B. melitensis vaccine strain M5-90 (identified as biovar 1) originated after a virulent B. melitensis (M28) isolation from a sheep was serially-passaged through chickens, treated with acriflavine and passaged for 90 generations in chicken embryo fibroblasts. This attenuated vaccine is considered to be one of the key factors that led to a rapid decline in the incidence of brucellosis in animals and humans in China during the 1970s to 1990s. Understanding the genetic similarities and differences between the vaccine strain and wild virulent strain will provide clues as to how the vaccine provides protection. To that end, we sequenced the B. melitensis parent strain M28 and the M5-90 strain using a whole-genome shotgun sequencing strategy. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with virulent strain M28 and other published genome sequences of two closely related strains B. melitensis 16M and ATCC 23457. We found 23 OCDs (ORFs with consistent differences) were different between the attenuated strains but they were identical amongst the virulent strains. A proteome study based on 2-D DIGE was performed in order to analyze the proteome difference between M28 and M5-90, which were grown under identical laboratory conditions. The majority of the 24 proteins heavily regulated at this stage were involved in bacterial metabolism, amino acid transportation, cell envelope and outer membrane biogenesis. This is the first genomic and proteomic analysis of an artificially attenuated Brucella strain. The results suggest that inactivation of genes involved in DNA/RNA metabolism, nutrient acquisition and utilization, and cell envelope or outer membrane proteins biogenesis may have played a role in the attenuation of M5-90.Compared with the M28 EF-Tu gene (BM28_A1261), M5-90 had one combined EF-Tu pseudogene with 50 SNPs and nine gaps (eight single nucleotide additions and one nucleotide deletion). To evaluate the potential role of EF-Tu in pathogenesis, the mutant tuf gene of B.melitensis (M28-tuf1, M28-tuf2 and M90-tuf2) was constructed by replacing the tuf gene of M28 and its attenuated M5-90 strain with kanamycin resistance gene. Double mutation recombinant was selected by adding kanamycin and ampicillin in the culture, and confirmed by PCR. The M28-tuf1 and M28-tuf2 mutant strains exhibited significant attenuation compared to M28 in mice infection model and still more pathogenically significant than M5-90. M28-tuf2 attenuation was compared to M28-tuf1, but the difference between the two groups is not considered statistically significant. These results indicated that the tuf-2 gene play an important role in the attenuation of M5-90. II. Immunologic mechanism of attenuation of M5-90 (murine transcriptome DGE analysis)Brucella species (spp.) reside in professional and non-professional phagocytes; in particular, macrophages are a major target in infected mammals. In comparison with other pathogenic bacteria, Brucella lack classical virulence factors, and their virulence is associated with their capacity to invade and propagate within host cells. The molecular mechanisms that cause M5-90 attenuation, particularly the alterations in pathogenicity between virulent strains of B. melitensis, remain poorly understood. In this study, we employed the Illumina Genome Analyzer platform to perform Digital Gene Expression (DGE) analysis of the peritoneal macrophage genome-wide transcriptome response to B. melitensis infection. A total of 1019 differentially expressed transcripts were identified in the macrophages four hours after infection with the different B. melitensis strains, with genes involved in the lysosome and SNARE interactions in vesicular transport being highly represented. Many proteins of the MAPK, Jak–STAT and TLR signaling pathways, along with the renal cell carcinoma and some other associated signaling pathways, were built in the protein–protein interaction networks (Signal-net). The key genes were some apoptosis-related genes, like Mapk3, Traf6, Chuk, Kras, Jak1 and Ptpn6. The regulation of these genes resulted in anti-apoptotic effects, especially M5-90 with weaker anti-apoptotic ability than M28. Our data provide new insight into the molecular attenuation mechanism of strain M5-90 and may enable vaccine residual virulence to be reduced, enhancing vaccine efficacy.