Atomic force spectroscopy of single adhesin on living Bordetella pertussis´s surface

L. ARNAL; N. CATTELAN; M. I. VILLALBA; M. F. CASTEZ; G. LONGO ; S. KASSAS; M. E. VELA; O. M. YANTORNO

Córdoba

Congreso; XI Congreso Argentino de Microbiología General; 2015

Sociedad Civil de Microbiología General (SAMIGE)

IntroductionPertussis is a human´s respiratory tract disease caused by mainly by Bordetella pertussis. Even after the introduction of vaccination, 60 years ago, pertussis remains a serious public health problem. Recent reports indicate that Bp might grow as biofilm attached to the upper respiratory tract as a mean to persist in the host and becoming an important risk of spread of these bacteria. Filamentous haemaglutinin (FHA) is the mayor adhesin of Bp and is involved in different steps of biofilm formation. Here, using Atomic Force Spectroscopy, we studied single interactions between purified FHA and a specific antibody attached to Si3Ni4 cantilevers and detected single FHA molecules in the surface of fixed and living Bp Tohama I reference strain cells. Experimental conditionsBp Tohama I (Pasteur Institute, France) and BpGR4 (BpFHA-, Bp Tohama derivative mutant lacking expression of FHA) strains were used through this study. Cultures were done in Erlenmeyer flaks in Stainer Sholter liquid medium, at 37ºC and under agitation (160 rpm). After 24 h of growth cells were harvested at 8000g for 5 min and incubated onto polyethyleneimine (PEI) coated glass slides (0,1% PEI in distillated water, overnight, 4ºC) for 1 h. For purified FHA, samples were prepared on muscovite mica (SPI v1 grade), by incubating 1% APTES solution for 1 min, dried with N2 current and incubating 30 µl of a 20nM solution of FHA in PBS for 15 min at room temperature and then washed with Milli-Q water three times and dried with N2. Samples were mounted on a NanoWizardII AFM´s liquid cell (JPK, Germany). Si3Ni4 cantilevers were modified with specific antibodies against FHA by linking them with glutaraldehyde 0.5%. Data were analyzed using OpenFovea software and JPK data processing software.Results and discussionA Force vs. log of loading rate curve was built and could be very well fitted with a linear function showing typical dependence of specific interactions with loading rate. Single cell spectroscopy was made at a scan rate of 500nm/s. At a first step we used fixed Bp Tohama I cells and several individual cells were mapped. Elasticity and force interaction maps were built from the recorded fv images, showing specific interactions on bacterial surface for wild type cells but not for FHA. We also tested the specificity of the interactions by making the force spectroscopy assay after incubating the cells with free antibody solution blocking the interaction´s sites. Results showed that proteins seem to be assembled into nanodomains or clusters of adhesins which also share location with the more rigid domains of the cell. The distribution of FHA in nanodomains could mean that the bacterium clusters FHA in certain regions of the membrane reaching biggest and strongest domains of interactions which could be advantageous for cell-substrate and cell- cell interactions during biofilm development.References 1)- Frits R. Mooi et all. Emerg Infect Dis. 2001, 7, 526-528.2)-Teemu Kallonen et all. Infection, Genetics and Evolution. 2011 Dec; 11(8):2034-42.3)- Matt S. Conover et all. Microbiology. 2010 Sep; 77(6):1439-55.4)- Julie H. Hannah et all. Infection and Immunity, Nov. 1994, p. 5010-5019.5)- Serra, D. O. et all. PLoS ONE 2011, 6, e28811.6)- Yersin A.et all. PNAS. 2003, jul 22 (15): 8736-41.7)- Charles Roduit et all. Nature Mothods 2012; 9, 774-775