Brucella enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent Brucella infection. We evaluated the importance of BtaF for respiratory Brucella infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with B. suis M1330 (wild type) and those receiving a Δ btaF mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. In vitro , these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against B. suis respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of B. suis was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with B. suis by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from B. suis respiratory infection, this adhesin constitutes a promising immunogen against mucosal B. suis infection.

A central aspect of Brucella pathogenicity is its ability to invade, survive, and replicate in diverse phagocytic and non-phagocytic cell types, leading to chronic infections and chronic inflammatory phenomena. Adhesion to the target cell is a critical first step in the invasion process. Several Brucella adhesins have been shown to mediate adhesion to cells, extracellular matrix components (ECM), or both. These include the sialic acid-binding proteins SP29 and SP41 (binding to erythrocytes and epithelial cells, respectively), the BigA and BigB proteins that contain an Ig-like domain (binding to cell adhesion molecules in epithelial cells), the monomeric autotransporters BmaA, BmaB, and BmaC (binding to ECM components, epithelial cells, osteoblasts, synoviocytes, and trophoblasts), the trimeric autotransporters BtaE and BtaF (binding to ECM components and epithelial cells) and Bp26 (binding to ECM components). An in vivo role has also been shown for the trimeric autotransporters, as deletion mutants display decreased colonization after oral and/or respiratory infection in mice, and it has also been suggested for BigA and BigB. Several adhesins have shown unipolar localization, suggesting that Brucella would express an adhesive pole. Adhesin-based vaccines may be useful to prevent brucellosis, as intranasal immunization in mice with BtaF conferred high levels of protection against oral challenge with B. suis.

Brucella spp. infection is frequently acquired through contaminated aerosols. The role of interleukin-1 beta (IL-1β) in the early pulmonary response to respiratory Brucella infection is unknown. As shown here, IL-1β levels in lung homogenates and bronchoalveolar lavage fluid (BALF) of mice intratracheally inoculated with B. abortus were increased at 3 and 7 days p.i. At 7 days p.i., pulmonary CFU numbers were higher in IL-1 receptor (IL-1R) knockout (KO) mice than in wild type (WT) mice. At different times p.i. CFU in lungs and BALF were higher in mice lacking some inflammasome components (caspase-1, AIM2, NLRP3) than in WT mice. At 2 days p.i. pulmonary levels of IL-1β and CXCL1 (neutrophils chemoattractant) were lower in caspase-1/11 KO mice. At day 3 p.i., neutrophils counts in BALF were lower in caspase-1/11 KO mice than in WT mice. During in vitro infections, IL-1β secretion was lower in alveolar macrophages from caspase-1/11, NLRP3 or AIM2 KO mice than in WT controls. Similarly, IL-1β production by B. abortus-infected alveolar epithelial cells was reduced by pretreatment with a specific caspase-1 inhibitor. This study shows that IL-1R, probably through IL-1β action, and the NLRP3 and AIM2 inflammasomes are involved in pulmonary innate immune protective mechanisms against respiratory B. abortus infection.