BP1492, a Bordetella pertussis exclusive diguanylate cyclase involved in biofilm formation and acidic stress response

J FERNÁNDEZ; F ZACCA; F SISTI

Salta

Congreso; Congreso PABMB-SAIB 2019; 2019

SAIB

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough. Despite the high vaccination coverage among children, pertussis is considered a re-emerging disease. Nearly all virulence factors in Bordetella pertussis are activated by a master two-component system, BvgAS. However other regulators systems are present in B. pertussis and needs further research. In the present work we focused on the role of the second messenger c-di-GMP, a key signaling molecule that regulates many important physiological processes in bacteria. Particularly we described for the first time a unique diguanylate cyclase (DGC), BP1492. DGC synthesizes c-di-GMP from two GTP molecules and are present in most bacterial genomes. BP1492 is present in all B. pertussis isolates sequenced to date and there are not homologues in other Bordetella species. Tipically, overexpresion of DGC in heterologous or homologous bacteria augments c-di-GMP intracellular concentration and enhanced biofilm formation. However after multiple efforts to express bp1492 from plasmid in different bacteria, DGC activity has not been evident for bp1492. BP1492 present a GAF domain in the N term portion. We speculated that a signal sensed by this domain may trigger DGC activity. This hypothetical signal may be absent in the experiments we did. To study the role of this DGC in B. pertussis we interrupted bp1492 gen and evaluate phenotypes previously described as c-di-GMP regulated in other bacteria.We evaluated biofilm formation over plastic surface with cristal violet technique. Wild type B. pertussis presented values similar to reported B. pertussis biofilms. However, the BP1492 mutant was enable to produce as high values as the wild type strain. Resistance to acidic stress is a phenotype regulated by c-di-GMP in other bacteria. During infection B. pertussis is exposed to acidic conditions when phagocyte by host cells. We speculate that c-di-GMP may regulate response to acid. To evaluate this, we exposed wild type and mutant strains to pH=4 and determinate viable bacteria after treatment. Interestingly, mutant strain presented a significant impaired survival at acidic pH. However, we did not observed differences in tolerance to oxidative stress induced by hydrogen peroxide. Overall, we described for the first time a B. pertussis exclusive DGC, BP1492. We showed that this DGC and thus the second messenger, c-di-GMP is involved in biofilm formation and resistance to acid in B. pertussis.