Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge

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Abstract
znuA is known to be an important factor for survival and normal growth under low Zn 2+ concentrations for Escherichia coli , Haemophilus spp., Neisseria gonorrhoeae , and Pasteurella multocida . We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus . Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 ( ΔznuA ) was constructed and found to be lethal in low-Zn 2+ medium. When used to infect macrophages, ΔznuA B. abortus showed minimal growth. Further study with ΔznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the ΔznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.