Characterization of Bordetella bronchiseptica diguanilate cyclase Bdcb.

SISTI F; BELHART, KEILA; FERNANDEZ, JULIETA

Congreso; PABMB-SAIB 2019; 2019

Bordetella bronchiseptica (Bb) is a gram negative respiratory pathogen that forms biofilm like structures in vivo. According to the signals it receives from the environment presents at least three phenotypically different phases: an avirulent phase, an intermediate phase, and a virulent phase. We previously showed that c-di-GMP regulates biofilm formation in B. bronchiseptica, like in other bacteria (Sisti et al. 2013). However, c-di-GMP may be involved in other stages required for effective infection and transmission. Bb can survive inside immune cells like macrophages. In order to determinate if c-di-GMP is involved in this process we evaluate Bb survival with deletions in multiple diguanylate cyclases. Deletion of bdcB (Bordetella diguanylate cyclase B) impaired bacterial survival inside macrophages. Four hours post infection, bacterial numbers in macrophages were below detection limits. Intracellular bacteria can resist macrophage bactericidal activity through different mechanisms. We evaluated B. bronchiseptica ΔbdcB (BbΔbdcB) resistance to acidic pH or oxidative burden (H2O2). While deletion of bdcB was not detrimental to H2O2 resistance, survival at pH lower than 5.0 was significantly affected. To further characterize BdcB, we overexpressed bdcB in Bb. We observed enhanced biofilm formation and inhibition of motility, as expected for an active diguanylate cyclase. This phenotypes were dependent on diguanilate cyclase (DGC) activity and the N-terminal domain, because an inactive version of BdcB (GGDEFxGGAAF) and an N-term-truncated BdcB did not inhibit motility, neither enhanced biofilm formation. Finally, we analyzed the expression of bdcB by a transcriptional fusion of the promoter to gfp in the three phenotypic phases of Bb. The bdcB expression was significantly higher in intermediate phase. We also determinated that bdcB expression is under repression of a response regulator required for bacterial resistance to oxidative stress and in vivo persistence, RisA (Jungnitz et al. 1998; Zimna et al. 2001). The present work represents other step on role of c-di-GMP comprehension in Bordetella pathogenesis and particularly the function of one of the ten diguanylate cyclases present in B. bronchiseptica genome in intracellular survival.