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.

Brucella, a Gram-negative bacterium with a high infective capacity and a wide spectrum of hosts in the animal world, is found in terrestrial and marine mammals, as well as amphibians. This broad spectrum of hosts is closely related to the non-classical virulence factors that allow this pathogen to establish its replicative niche, colonizing epithelial and immune system cells, evading the host’s defenses and defensive response. While motility is the primary role of the flagellum in most bacteria, in Brucella, the flagellum is involved in virulence, infectivity, cell growth, and biofilm formation, all of which are very important facts in a bacterium that to date has been described as a non-motile organism. Evidence of the expression of these flagellar proteins that are present in Brucella makes it possible to hypothesize certain evolutionary aspects as to where a free-living bacterium eventually acquired genetic material from environmental microorganisms, including flagellar genes, conferring on it the ability to reach other hosts (mammals), and, under selective pressure from the environment, can express these genes, helping it to evade the immune response. This review summarizes relevant aspects of the presence of flagellar proteins and puts into context their relevance in certain functions associated with the infective process. The study of these flagellar genes gives the genus Brucella a very high infectious versatility, placing it among the main organisms in urgent need of study, as it is linked to human health by direct contact with farm animals and by eventual transmission to the general population, where flagellar genes and proteins are of great relevance.

Shigellosis is a diarrheal disease and the World Health Organization prompts the development of a vaccine against Shigella flexneri. The autotransporters SigA, Pic and Sap are conserved among Shigella spp. We previously designed an in silico vaccine with immunodominat epitopes from those autotransporters, and the GroEL protein of S. typhi as an adjuvant. Here, we evaluated the immunogenicity and protective efficacy of the chimeric multiepitope protein, named rMESF, in mice against lethal infection with S. flexneri. rMESF was administered to mice alone through the intranasal (i.n.) route or accompanied with Complete Freund’s adjuvant (CFA) intradermically (i.d.), subcutaneously (s.c.), and intramuscular (i.m.), as well as with Imject alum (i.m.). All immunized mice increased IgG, IgG1, IgG2a, IgA and fecal IgA titers compared to PBS+CFA and PBS+alum control groups. Furthermore, i.n. immunization of mice with rMESF alone presented the highest titers of serum and fecal IgA. Cytokine levels (IFN-γ, TNF-α, IL-4, and IL-17) and lymphocyte proliferation increased in all experimental groups, with the highest lymphoproliferative response in i.n. mice immunized with rMESF alone, which presented 100% protection against S. flexneri. In summary, this vaccine vests protective immunity and highlights the importance of mucosal immunity activation for the elimination of S. flexneri.

As an alternative brucellosis prevention method, we evaluated the immunogenicity induced by new multivalent DNA vaccines in BALB/c mice. We constructed the vaccines by fusion of BAB1_0273 and/or BAB1_0278 open reading frames (ORFs) from genomic island 3 (GI-3) and the Brucella abortus 2308 sodC gene with a link based on prolines and alanines (pV273-sod, pV278-sod, and pV273-278-sod, resp.). Results show that immunization with all tested multivalent DNA vaccines induced a specific humoral and cellular immune response. These novel multivalent vaccines significantly increased the production of IgM, IgG, and IgG2a antibodies as well as IFN-γ levels and the lymphoproliferative response of splenocytes. Although immunization with these multivalent vaccines induced a typical T-helper 1- (Th1-) dominated immune response, such immunogenicity conferred low protection levels in mice challenged with the B. abortus 2308 pathogenic strain. Our results demonstrated that the expression of BAB1_0273 and/or BABl_0278 antigens conjugated to SOD protein can polarize mice immunity to a Th1-type phenotype, conferring low levels of protection.

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.

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 of a Zinc-dependent metalloproteinase. Here, its contribution in the process of intracellular adaptation was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this Zinc-dependent metalloproteinase is a cytoplasmic protein that conforms an operon with a transcriptional regulator, which may constitute a type II toxin-antitoxin system. Functionally, this Zinc-dependent metalloproteinase participated neither in the adherence nor 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, reducing both its co-localization with calnexin, present in endoplasmic reticulum derived vesicles, and its intracellular replication within macrophages. Besides, B. abortus Δ270-infected macrophages produced significantly higher levels of TNF-α, IL-6, CD80 and CD86 than B. abortus 2308, even when several genes involved in virulence (vjbR, hutC, bvrR, virB1) were up-regulated in this mutant. Finally, its deletion significantly reduced the capacity of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, we discuss the role of this Zinc-dependent metalloproteinase in the regulation of the virulence of this pathogen, concluding that it contributes significantly to the intracellular adaptation of B. abortus 2308 during the infection of macrophages.Author summaryBrucella abortus is the causative agent of the brucellosis, a highly contagious diseases. A Zinc-dependent metalloproteinase contributes significantly in the intracellular survival. Here, we demonstrate that this metalloproteinase has homology with ImmA/IrrE proteases, which are involved in the bacterial resistance to hostile environment. Furthermore, it conforms a gene pair with a transcriptional regulator, being required by B. abortus to escape from phagolysosomes, to achieve the endoplasmic reticulum and replicate within macrophages. Its deletion from B. abortus stimulated the macrophages, which produced higher levels of pro-inflammatory cytokines and co-stimulatory proteins. This pathogen showed a reduced ability to adapt and grow under acidic conditions, which would negatively affect its escape from phagolysosomes and consequently, stimulating macrophages. Therefore, this work describes how this Zinc-dependent metalloproteinase significantly contributes in the intracellular adaptation of B. abortus 2308 in macrophages.