Distribution of the surface-associated adhesin Filamentous Haemagglutin on Bordetella pertussis living cells using Atomic Force Spectroscopy

ARNAL, LAURA; CATTELÁN, NATALIA; CASTEZ, FEDERICO; ALVAREZ ACOSTA, ANABEL; VILLALBA, MARÍA INÉS; QUINTANA VÁZQUEZ, DIÓGENES; GUILLÉN NIETO, GERARDO; VELA, MARÍA ELENA; YANTORNO, OSVALDO M.

Varadero, Meliá Marina Varadero Hotel, Cuba

Congreso; First Internacional Convention Immunopharmacology- Vaccipharma 2015; 2015

International Union of Basic and Clinical Pharmacology (IUPHAR) and the Cuban Society of Pharmacology

Introduction. Filamentous hemagglutinin (FHA) is a surface-associated adherence protein of Bordetella pertussis, component of acellular pertussis vaccines. The localization of FHA protein to the cell surface is a key step required by Bp to irreversibly attach to a surface and form a biofilm. Here we conducted experiments to determine the location and dynamical behavior of FHA by using Atomic Force Microscopy (AFM). Methods. Bp Tohama I reference strain, an avirulent phase mutant, and a BpFHA− a Bp Tohama derivative mutant lacking expression of FHA, were used. Cells from solid medium were fixed and mounted on a NanoWizardII AFM´s liquid cell (JPK, Germany). Si3Ni4 cantilevers were modified with specific antibodies against FHA. Force volumes images were made on each sample. Data were analyzed using OpenFovea software and JPK data processing software. Maps were made using homemade software.Results. FHA location in living cells was studied by nano-mechanical analysis. The wild type strain showed higher Young modulus compared to the two mutant strains. The increase was associated to the presence of FHA protein, and more specifically values over 0,55 MPa were associated to locations where the protein is exposed. Young modulus maps showed a non homogeneous distribution of FHA on the cell surface but rather nanodomains were the proteins might be expressed. These findings suggest a collaborative behaviour among adhesins to strongly adhere to surfaces and could represent an advantage during host-pathogen interaction. Experiments using an antibody functionalized probe to recognize FHA proteins showed a most probable value for the antibody-FHA interaction around 150 nN and an increase in the recognition events after the application of the external force that ranged between 10 and 24% per cell was obtained. Conclusions. FHA protein have a dynamical behaviour on the surface of living cells that show a collaborative mechanism in response to external stimuli.