DELETION OF PURE ATTENUATES BRUCELLA-MELITENSIS 16M FOR GROWTH IN HUMAN MONOCYTE-DERIVED MACROPHAGES

We constructed a defined purine-auxotrophic mutant of Brucella melitensis 16M by chromosomal gene replacement, We electroporated B, melitensis 16M with suicide plasmids containing a kanamycin resistance cassette that replaced 226 bp at the carboxyl end of purE, the intergenic region, and 18 bases of the purK open reading frame, Recombinant B, melitensis Delta purE201 required exogenous purines for growth on minimal media. Purine auxotrophy was complemented by electroporation of B, melitensis Delta purE201 with a plasmid, pSD5, carrying only the wild-type purE gene, In in vitro assays of virulence, B, melitensis Delta purE201 failed to grow in human monocyte-derived macrophages, while the growth of wild-type 16M and the complemented strain, Delta purE201(pSD5), increased by nearly two logs, These results suggest that B, melitensis Delta purE201 will be attenuated in animals and humans and thus may be useful as a live attenuated vaccine.