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

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 (⌬znuA) was constructed and found to be lethal in low-Zn 2؉ medium. When used to infect macrophages, ⌬znuA B. abortus showed minimal growth. Further study with ⌬znuA 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 ⌬znuA 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. Zn2ϩ is an essential mineral required by bacteria as either a structural or catalytic cofactor (32). Bacterial survival and proliferation in the environment and within animal hosts are critically dependent on the uptake and sequestration of transition metals, such as Zn 2ϩ (4). This is problematic, because free Zn 2ϩ concentrations in mammalian hosts are very low, so as to prevent bacterial colonization. To acquire the necessary Zn 2ϩ for its metabolism, bacteria have evolved several types of proteins that are involved in binding and transporting zinc (9).The translation products of the znuABC operon found in Escherichia coli (5, 31), Haemophilus spp. (27), Neisseria gonorrhoeae (8), Pasteurella multocida (15), and Synechocystis sp. strain 6803 (4) constitute a high-affinity periplasmic binding protein-dependent and ATP-binding cassette (ABC) transport system for Zn 2ϩ . In gram-negative bacteria, ABC transporters are involved in the active transport of molecules from periplasm to the cytosol (19). In addition, znuA mutants in H. ducreyi (27) and P. multocida (15) were found to be significantly less virulent than wild-type strains when tested in animal models.B. abortus is a gram-negative facultative, intracellular pathogen capable of infecting both wildlife and livestock (7), and it is able to cause severe zoonotic infection in humans (3, 34). Currently, there are no human Brucella vaccines, and current livestock vaccines such as S19 and RB51 are virulent in humans. Attempts to develop live brucellae vaccines have met with varied success. For instance, inactivation of the amino acid biosynthesis pathway genes pheA, trpB, and dagA displayed little or no attenuation in cultured murine macrophages or in mice (1). The mutants of purine biosynthesis pathway genes purL, purD, and purE (1) displayed significant attenuation in BALB/c mice, but live brucellae remained viable after 12 weeks, suggesting that their virulence was not sufficiently attenuated for adoption as...

A Novel Zinc Binding System, ZevAB, Is Critical for Survival of Nontypeable Haemophilus influenzae in a Murine Lung Infection Model

“…Comparison of our list of potential DsbA substrates to the results of HITS revealed three genes that fit these criteria: znuA (s.i., 0.101), encoding the periplasmic component of a high-affinity zinc uptake transporter (39, 53), nlpD (s.i., 0.000), encoding a lipoprotein suggested to be involved in daughter cell separation (64,65), and HI1249 (s.i., 0.106), encoding a putative protein herein referred to as ZevA. The role of znuA in virulence has been studied by several groups (3,11,14,21,36,57,75), and nlpD has been implicated in the virulence of Yersinia pestis and Yersinia enterocolitica (12,64). However, zevA has not been previously investigated and its role in virulence is unknown.…”

“…Here, we generated a list of potential substrates of the periplasmic disulfide oxidoreductase protein DsbA (see Table S2 in the supplemental material) and integrated our findings with the results of a previous study in which HITS technology was used to identify genes required for lung infection (22). Using this strategy, we identified three genes that were potentially important for lung infection, including two, znuA and nlpD, that had previously been implicated in the virulence of several organisms (3,11,12,14,21,36,57,64,75), and a third gene, zevA, whose function had not been determined. Therefore, zevA and its neighboring gene, zevB, were investigated for their roles in the pathogenesis of a clinical NTHI isolate.…”

Znu Is the Predominant Zinc Importer in Yersinia pestis during In Vitro Growth but Is Not Essential for Virulence

“…In recent years, the virulence of a variety of bacterial pathogens has been shown to be dependent upon Zn acquisition from the host via Znu or related C9 ABC transporters (1,15,24,42,57,64,67,74,90,104,107). Experiments were undertaken, therefore, to determine whether the Znu system is required for the virulence of Y. pestis.…”

“…Although most SBPs appear to have specificity for a single metal ligand, there is in vitro and in vivo experimental evidence that some non-Znu C9 SBPs possess specificities for multiple metals. The relevance of Zn uptake and the Znu system in particular is highlighted by a number of reports demonstrating that pathogens lacking this transporter are markedly attenuated in virulence (1,15,24,42,57,64,67,74,90,104,107).…”

Desenvolvimento e avaliação de uma cepa knockout de Brucella abortus obtida pela deleção do gene virB10

“…(Hartigh et al 2004). Yang et al (2006) relataram que a cepa mutante ΔznuA foi atenuada em camundongos BALB/c. O gene znuA expressa uma proteína relacionada ao sistema de transporte de Zn +2 , que é um mineral essencial para a bactéria com função estrutural e como cofator catalítico.…”