Cloning, Expression, Purification and Characterization of Brucella abortus Auto-transporter Genes and Their Products

Jeffres, Nolan
Brucellosis is an economically important cause of abortions in livestock and wildlife worldwide caused by the facultative intracellular pathogen Brucella abortus. One understudied molecular mechanism of in-vivo survival used by B. abortus is adherence to target cells by Type-V (auto-transporting) proteins. BruAb1_1988 and BruAb1_1989 are two such proteins that have been identified within a Cluster of Orthologous Groups (COGs) of proteins expressed in-vivo during infection. Attempts to express BruAb1_1989 have been unsuccessful. A bioinformatics approach, including a 3-D structural analysis, has predicted that it is likely a multimeric inner membrane protein composed of primarily two (folded) α-helical domains. BruAb1_1988 has been expressed through traditional cloning methods, in two segments separating the passenger and β-barrel domains. Western blots conducted on the domains using NVSL Bovine check serum for B. abortus haven't yielded any positive results. This result isn't entirely unexpected, as the gene for BruAb1_1988 should produce a truncated product in B. abortus. The ortholog in B. melitensis however is expected to produce a functional protein. Because of this future characterization will continue, including further characterization of the β-barrel domain because of its potential use in identifying other in-vivo expressed autotransporters. Further understanding of the structural and functional nature of these and other Brucella Type-V proteins will potentially lead to their use in a next-generation diagnostic assay that separates B. abortus and B. melitensis, another clinically relevant species contributing to Brucellosis outside of the United States.
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