CONICET | Buscador de Institutos y Recursos Humanos

Bordetella bronchiseptica (Bb) is a gram-negative bacterium that cause respiratory diseases in different animals. We focus on the study of a bacterial regulatory system meditated by the cyclic di-GMP (cdG) that is a second messenger synthesized by diguanylate cyclases and degraded by phosphodiesterases. We previously showed that cdG regulates motility and biofilm formation in Bb, like in other bacteria. However, the role of cdG during infection, pathogenesis and transmission is still unclear. In this work we describe the diguanylate cyclase BdcB (Bordetella diguanylate cyclase B) as an active diguanylate cyclase that can increase the biofilm formation and inhibit motility in Bb. Furthermore, we analyze the role of BdcB in Bb intracellular survival inside macrophages obtained from the bone marrow of BALB/c mice. While the wild type (wt) was recovered from macrophages after 24 hours, bdcb null-mutant (∆bdcB) was undetectable at four hours post infection. To determine if macrophages were killing bacteria or bacteria were killing macrophages, we performed a dynamic assay with propidium iodide (PI). PI binds to DNA from dead cells. In this way, macrophage death can be measured according to the increase in fluorescence generated by the binding of PI to DNA. We observed that the fluorescence was higher in macrophages in contact with ∆bdcB, indicating that ∆bdcB was killing the cells faster than the wt strain. In addition, we confirmed that ∆bdcB is more cytotoxic by measuring lactate dehydrogenase (LDH) release: LDH release was 50% for wt and 70 % for ∆bdcB. We also evaluated the cytokine secretion profile from the supernatants of infected macrophages and an increase in interleukins IL-10 and IL-6 was observed in ∆bdcB compared to the wt strain. With these results, we decided to evaluate by qRT-PCR the expression the different virulent factors involve in Bb pathogenesis (adenylate cyclase, filamentous haemagglutinin, pertactin, dermonecrotic toxin) and genes of the type three secretion system (T3SS). We found no differences in the expression of the virulence factors but the expression of many genes of the T3SS was increased in ∆bdcB. This result was confirmed by western blot, indicating that BdcB negatively regulates the expression of the T3SS in Bb. Finally, there were no difference in upper respiratory tract colonization in BALB/c mice when they were infected with 105 CFU of wt or ∆bdcB strain. However, the production of different cytokines such as IL1α and TNFα and chemokines MIP-1β, BLC, RANTES and MDC were higher in lungs of mice infected with ∆bdcB at day 7 post-infection. The present work represents a new step in the understanding of the role of c-diGMP in Bb pathogenesis and particularly the function of one of the ten diguanylate cyclases present in Bb genome. Understand the interaction of bacteria with cells of the immune system during the infection is important to contain and control the spread of Bordetella-caused disease.