Unraveling the Role of the Zinc-Dependent Metalloproteinase/HTH-Xre Toxin/Antitoxin (TA) System of Brucella abortus in the Oxidative Stress Response: Insights into the Stress Response and Virulence.

Toxin/antitoxin (TA) systems have been scarcely studied in Brucella abortus, the causative agent of brucellosis, which is one of the most prevalent zoonotic diseases worldwide. In this study, the roles of a putative type II TA system composed by a Zinc-dependent metalloproteinase (ZnMP) and a transcriptional regulator HTH-Xre were evaluated. The deletion of the open reading frame (ORF) BAB1_0270, coding for ZnMP, used to produce a mutant strain, allowed us to evaluate the survival and gene expression of B. abortus 2308 under oxidative conditions. Our results showed that the B. abortus mutant strain exhibited a significantly reduced capacity to survive under hydrogen peroxide-induced oxidative stress. Furthermore, this mutant strain showed a decreased expression of genes coding for catalase (katE), alkyl hydroperoxide reductase (ahpC) and transcriptional regulators (oxyR and oxyR-like), as well as genes involved in the general stress response, phyR and rpoE1, when compared to the wild-type strain. These findings suggest that this type II ZnMP/HTH-Xre TA system is required by B. abortus to resist oxidative stress. Additionally, previous evidence has demonstrated that this ZnMP also participates in the acidic stress resistance and virulence of B. abortus 2308. Therefore, we propose a hypothetical regulatory function for this ZnMP/HTH-Xre TA system, providing insight into the stress response and its potential roles in the pathogenesis of B. abortus.

A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages.

Brucella abortus is a pathogen that survives in macrophages. Several virulence factors participate in this process, including the open reading frame (ORF) BAB1_0270 codifying for a zinc-dependent metalloproteinase (ZnMP). Here, its contribution in the intracellular adaptation of B. abortus was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this ZnMP did not participated in either the adherence or the initial intracellular traffic of B. abortus in macrophages. Nevertheless, its deletion significantly increased the co-localization of B. abortus Δ270 with phagolysosomal cathepsin D and reduced its co-localization with calnexin present in endoplasmic reticulum (RE)-derived vesicles. Although B. abortus Δ270 showed an upregulated expression of genes involved in virulence (vjbR, hutC, bvrR, virB1), it was insufficient to reach a successful intracellular replication within macrophages. Furthermore, its attenuation favored in macrophages infected the production of high levels of cytokines (TNF-α and IL-6) and co-stimulatory proteins (CD80 and CD86), signals required in T cell activation. Finally, its deletion significantly reduced the ability of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, and considering that this ZnMP has homology with ImmA/IrrE proteases, we discuss its role in the virulence of this pathogen, concluding that ZnMP is required in the intracellular adaptation of B. abortus 2308 during the infection of macrophages.

Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice.

Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis.