The diguanylate cyclase BdcB is involved in Bordetella bronchiseptica intracellular survival

BELHART, KEILA; HARVILL, ERIC T.; TAYLOR, DAWN; FERNANDEZ, JULIETA; CARTELLE, MÓNICA; SISTI, FEDERICO

Berlin

Simposio; International Symposium: Nucleotide Second Messenger Signaling in Bacteria; 2018

Bordetella bronchiseptica is a gram negative respiratory pathogen that forms biofilm like structures in vivo. We previously showed that c-di-GMP regulates biofilm formation in B. bronchiseptica, like in other bacteria1. However, c-di-GMP may be involved in other steps required for effective infection and transmission.B. bronchiseptica can survive inside immune cells like macrophages. In order to determinate if c-di-GMP is involved in this process we evaluate survival of B. bronchiseptica with deletions in 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. Sequence analyses of BdcB predicted a bona fide diguanylate cyclase located in the cytoplasm. Thus BdcB, can complement a diguanylate cyclase defective Pseudomonas fluorescens biofilm deficient mutant. Also, biofilm formation was enhanced when BdcB was over-expressed in B. bronchiseptica, as expected for an active diguanylate cyclase.Intracellular bacteria can resist macrophage bactericidal activity through different mechanisms. We evaluated B. bronchiseptica ∆bdcB (Bb∆bdcB) resistance to acidic pH or oxidative burden (H2O2). While deletion of bdcB is not detrimental to H2O2 resistance, survival at pH lower than 5.5 was significantly affected.Interestingly, deletion of bdcB did not affect biofilm formation in vitro in standard growth media. However, biofilm formation at pH 5.5 by Bb∆bdcB was higher than wild type.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. Understanding the nature of intracellular survival during infection is important to contain and control the spread of Bordetella-caused disease.1. Sisti F. et al.(2013). Microbiology. 2013 May;159(Pt 5):869-79.