1. Lap proteins are a c-di-gmp effector system that controls biofilm formation in Bordetella bronchiseptica

SISTI F; AMBROSIS N; FERNANDEZ J; O´TOOLE G

Buenos Aires

Congreso; 11th International Bordetella Symposium; 2016

no hay una institución organizadora

Objectives. We have previously demonstrated that a cyclic di-GMP-dependent mechanism is involved in biofilm formation in Bordetella bronchiseptica. We identified three candidates that could be part of the system. Those proteins were first identified on Pseudomonas fluorescens and named Lap´s (for large adhesion proteins). The purpose of the present study is to characterize Lap proteins (LapD, LapG and LapA) in Bordetella. Materials and Methods. Deletion of lapA and lapG genes were genetically engineered rendering BblapA and BblapG respectively. Biofilm formation was evaluated by Cristal Violet (CV) assays over PVC microplate dishes and glass tubes. We also performed static culture experiments to analyze biofilm structures by fluorescence and electronic microscopy. To evaluate the role of Lap proteins during the respiratory infection period we intranasally infected four to six week-old female BALB/c mice, recovered and count UFC/ml of nasal cavity and lungs.Results. Accordingly with our proposed model, CV experiments showed that BblapG has enhanced biofilm formation ability while BblapA has lower capability to develop biofilm over PVC and glass. In addition, microscopy experiments confirmed those results. Overexpression of LapG reduced biofilm formation. Significantly higher numbers of BblapG compare to wild type infected mice were observed 14 days post infection. Conclusion. Our findings demonstrate that Lap proteins are involved in biofilm formation in B. bronchiseptica. Moreover, we observed that LapG cleaved LapA. Similar phenotypes observed in lapA mutant and overexpressing lapG strain support the hypothesis that LapG regulates LapA presence on the bacterial surface. Interestingly, deletion of lapG enhance B. bronchiseptica infection in lungs. This project shed light on the understanding of a very important pathogenic characteristic that is biofilm formation.