LapG protein controls biofilm formation in Bordetella bronchiseptica

AMBROSIS, NICOLÁS MARTÍN; SISTI, FEDERICO; FERNANDEZ, JULIETA

Rosario

Congreso; IX CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2013

Bordetella bronchiseptica is a pathogenic bacterium that causes respiratory infections in a wide variety of host. Like otherpathogens forms biofilm-like structures on abiotic surfaces. Different authors even observed these structures in vivo in the nasalepithelium of mice infected with B. bronchiseptica. We have made progress in analyzing the effect of changes in the intracellularconcentration of the second messenger c-di-GMP on biofilm formation of B. bronchiseptica. Particularly we determined thatoverexpression of a diguanylate cyclase induced in B. bronchiseptica increased ability to form biofilm. However, the exactmechanism of this process and the bacterial factors involved in biofilm formation has not been yet determined.In Pseudomonas fluorescens a c-di-GMP effectors system controls biofilm formation by inside-out signaling and surface proteincleavage. Different proteins designed leucin aminopeptidase (Lap) proteins are involved. LapA is a surface adhesin located inthe outer membrane. LapA binding to the cell surface is controlled by LapD, which can bind c-di-GMP through its domainphosphodiesterase. In P. fluoresens when levels of c-di-GMP are high, the binding of c-di-GMP to LapD promotes the formationof biofilm through LapA accumulation on the cell surface. LapG is a periplasmic calcium-dependent cysteine protease thatcleaves cell surface LapA, freeing the adhesin and preventing biofilm formation. Accordingly, when LapG is deleted from P.fluorescens genome biofilm formation is enhanced. On the other hand, when LapG is overexpressed biofilm is prevented.In the present work, homology to LapD, LapG and LapA was observed with B. bronchiseptica proteins BB1184, BB1185 andBB1186 respectively. To evaluate functionality, BB1185 (LapGBb) gene from B. bronchiseptica was amplified and cloned in thereplicative plasmid pMQ72 and transformed in P. fluorescens with deleted lapG. Biofilm formation was evaluated in therecombinant bacteria and as expected biofilm development was impaired when LapGBb was expressed, corroboratingfunctionality of this protein.In order to evaluate if LapGBb was involved in biofilm formation lapGBb was subcloned to appropriate expression plasmid forBordetellae, pBBR1MCS-5 to induce overexpression in a wild type background. The resulting strain Bb-LapGBb producedsignificantly less biofilm than parental strain on abiotic surface when was determined by the cristal violet method. Surprisinglywhen standard growth media Stainer-Scholte was supplemented with calcium in the millimolar range biofilm formation wassignificantly enhanced in all strains tested. In these conditions Bb-LapGBb biofilm levels were also below those observed for wildtype strain corroborating that LapGBb is involved in biofilm development. Further studies are in process to determinate if Lapsystem is important to biofilm development in vivo.