IBBM   21076
INSTITUTO DE BIOTECNOLOGIA Y BIOLOGIA MOLECULAR
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
CHARACTERIZATION OF BORDETELLA BRONCHISEPTICA DIGUANILATE CYCLASE BdcB
Autor/es:
BELHART, KEILA; FERNANDEZ, JULIETA; SISTI, FEDERICO
Lugar:
Salta
Reunión:
Congreso; LV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2019
Institución organizadora:
SAIB y PABMB
Resumen:
Bordetella bronchiseptica (Bb) is a gram negative respiratory pathogen that forms biofilm like structures in vivo. According to the signals itreceives from the environment presents at least three phenotypically different phases: an avirulent phase, an intermediate phase, and a virulentphase. 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 likemacrophages. In order to determinate if c-di-GMP is involved in this process we evaluate Bb survival with deletions in multiple diguanylatecyclases. 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 throughdifferent mechanisms. We evaluated B. bronchiseptica ∆bdcB (Bb∆bdcB) resistance to acidic pH or oxidative burden (H2O2). While deletion ofbdcB was not detrimental to H2O2resistance, survival at pH lower than 5.0 was significantly affected. To further characterize BdcB, weoverexpressed 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 theexpression of bdcB by a transcriptional fusion of the promoter to gfp in the three phenotypic phases of Bb. The bdcB expression wassignificantly higher in intermediate phase. We also determinated that bdcB expression is under repression of a response regulator required forbacterial resistance to oxidative stress and in vivo persistence, RisA (Jungnitz et al. 1998; Zimna et al. 2001). The present work represents otherstep on role of c-di-GMP comprehension in Bordetella pathogenesis and particularly the function of one of the ten diguanylate cyclases presentin B. bronchiseptica genome in intracellular survival.