Genomic and Proteomic Analyses of Brucella melitensis VRI 6530/11 (Malaysian Isolate)

Brucellosis is a bacterial zoonotic disease that affects a variety of domestic and wildlife animals. In Malaysia, data on bacteriological and serological studies of brucellosis has provided evidence that the most prevalent species is Brucella melitensis. The disease constitutes a significant hurdle in the development of the small ruminant industry. It also contributes to unfavorable socioeconomic and public health impacts. The Department of Veterinary Services (DVS) Malaysia employed a test and slaughter strategy to eradicate the disease. Despite employing this strategy, the disease is still prevalent in the country. Hence, there is an urgent need for better control measures, which require a deeper understanding of the biology of the B. melitensis isolated in Malaysia. Whole genome sequencing and proteomic analyses are important initial steps in understanding the biology of B. melitensis local isolate. The objectives of this study are to: characterize the genome of B. melitensis VRI 6530/11 isolate and determine the antigenicity of the outer membrane proteins (OMPs) and recombinant OMP31 of the isolate. The whole genome was sequenced using illumina HiSeq platform with the insert size of ~200 bp. The potential virulence genes were identified by Basic Local Alignment Search Tool (BLAST) against pathogen host interactions (PHI) and virulence factor database (VFDB). All the genes were functionally annotated by BLAST against non-redundant, Swiss-Prot protein and Kyoto Encyclopedia of Genes and Genomes databases. The OMPs were extracted and characterized using Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE), while their antigenicity was tested using western blotting, modified indirect ELISA and developed rapid slide agglutination test. The omp31 gene was amplified, cloned in pET-24d(+) and expressed in E. coli BL21. Subsequently, the antigenicity of recombinant OMP31 was evaluated using western blotting against the sera of immunized rabbit and an infected goat. The final genome assembly of B. melitensis VRI 6530/11 had 33 scaffolds and 44 contigs with total scaffolds size of ~3.28 Mb. A total of 3,238 protein coding genes were predicted to be from the genome. A total of 14 genes were identified as potential virulence factors, based on the PHI database. The BLAST results against VFDB revealed 52 genes were involved in: i) immune evasion which include lipopolysaccharides (LPS), ii) intracellular survival which include cyclic β-1,2 glican (CβG), iii) two component system which include BvrR/BvrS, iv) type IV secretion system (T4SS) and v) iron uptake called brucebactin. Gene ontology revealed that 783 genes were involved in the outer membrane proteins and 75% percent out of these genes have been annotated. The OMPs profile showed a unique protein band of molecular weights of 5, 8, 23, 56, 60 and 110 kDa. The OMPs and recombinant OMP31 have high antigenicity in detecting antibodies against Brucella. Thus the OMPs can be used as antigens for serological diagnosis of brucellosis in goats. The genomic data has provided new insights into virulence mechanisms and pathogenesis of B. melitensis VRI 6530/11 isolate. The outcome of this study will benefit future research in developing subunit vaccine to control B. melitensis infection