High resolution visualization and characterization of cell surface adhesion protein in three-dimensional nanoscale interphases

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Reynaldo T. Diaz (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Dennis LaJeunesse

Abstract: This dissertation studies microbial adhesion to surfaces using high magnification scanning particle beam microscopy. To study the interaction with the wings of cicada insects as our main surface of interest, the microbial model Saccharomyces cerevisiae with various levels of adhesive protein was used. Upon magnifying the cicada insect’s wing one hundred thousand times, it evidences that the surface is composed of an even array of tiny nails. The past research as well as our group previous work have shown that the nanostructure surfaces like the cicada wing trigger responses in microbes that can range from cell proliferation to total cell-wall disruption. However, up to this day the reasons why certain nanostructured surfaces disrupt the cell-wall remained unclear. The central hypothesis of this thesis is to investigate the role of adhesion protein FLO11p found in S. cerevisiae cell wall and the surface topography at the nanoscale using scanning particle beam microscopy as the main technique. A first step towards proving the central hypothesis was the development and use of immunogold labelling of FLO11p technique to be used with the scanning particle beam microscope and dispersive x-ray spectrometry. The image generated by secondary electrons and backscattered electrons has been traditionally used as correlative microscopy. However, incorporating electron energy dispersive x-ray spectrometry (EDS) is advantageous for elemental confirmation. Using immunogold SEM EDS allowed us to ratify that our studies of FLO11p and surface adhesion was not a result of artifacts of electron microscopy. We show that the cell strains with normal levels of FLO11p and knockout levels of FLO11p are unable to withstand the penetration of the 100-nm tall nanopillars, while the strain that overexpresses FLO11p could withstand the effects of the nanopillars. This was an unexpected finding as we predicted a linear response of deleterious effect to the expression levels of FLO11p. Findings of this study highlight the importance of surface topography when designing surfaces for microbial control, where surfaces can selectively promote or kill microbes based on their topographical features.

Additional Information

Language: English
Date: 2018
Cicada, FLO11, HIM, Immunogold, Microscopy, SEM
Saccharomyces cerevisiae $x Biotechnology
Microorganisms $x Adhesion
Microbial proteins
Immunogold labeling
X-ray spectroscopy

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