Microcontact Printing of Thiols: Changing the Way Cell Attachment is Investigated
B. Leite , 1 * R. Dziedzic , 2 L. Cruz , 3 A. L. Gillian-Daniel , 2 C. Nielsen , 1 and L. De La Fuente 3 1 JEOL USA, Inc. , 11 Deaborn Road , Peabody , MA 01960 2 University of Wisconsin , MRSEC Education Group , 1415 Engineering Drive , Madison , WI 53706 3 Auburn University , Plant Pathology , 209 Rouse Life Sciences Bldg. , Auburn , AL 36849
*
bleite@jeol.com Introduction
Biofilms are complex three-dimensional structures formed by microorganisms. Biofilms protect microorganisms (for example, pathogenic bacteria) against antimicrobial compounds, therefore posing a threat to human, animal, and plant health. Despite numerous biofilm formation studies, details of factors influencing bacterial cell-to-cell and cell-surface interaction mechanisms are often unclear. We have been observing the unique association of disciplines and new techniques dedicated to examining how living and non-living entities adhere or detach to/from particular substrates. There are a number of mechanical effects, physical forces, and chemical changes that are critical in these interac- tions [ 1 ]. The importance of this cell attachment/adhesion phenomenon is grounded in the fact that bacterial pathogens will alter their physiological-biochemical and molecular status, once anchorage has been established, by activating pathogenicity factors. Specific connections to host surfaces at a molecular level seem to be a critical first step. Bacterial cells attach to surfaces, above all, because it is a survival mechanism. Pathogenic bacteria will attach to surfaces to more effectively colonize the host, thus providing them a competitive advantage over unattached cells. That is why attraction of human pathogens to medical instruments, tubing, and connectors is currently being investigated by several research groups. Not surprisingly, xylem-limited plant pathogenic bacteria are considered successful if they are able to establish themselves on the xylem walls in numbers that help them achieve massive growth [ 2 ]. Cell attachment to non-host surfaces is also a survival strategy, allowing the pathogen to wait for an appropriate opportunity to be transferred to a host organism. For instance, fresh produce can carry food-borne pathogens without causing any disease to the plant. In this case, the bacterial cells are attached to produce surfaces until bacterial cells meet an ideal environment (host) to thrive. Once attachment is established, it is almost impossible to eliminate these contaminants by regular washing, exposing us to danger even when we follow proper food processing recommendations [ 1 ].
For the present work, we tested the xylem-limited colonizing plant pathogen Xylella fastidiosa [ 3 ]. T ese bacterial cells are able to attach themselves to the internal walls of xylem vessels and form colossal amounts of biofi lm, eventually clogging these vessels and killing the host by restricting access to water and nutrients. T e disorder is known as Pierce’s disease in grapevines and mainly aff ects California wine industry, though it is present in most of the southern areas of the US.
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T e objectives of the present work are: (1) to demonstrate a new and less time-consuming approach for studying biofi lm formation and cell attachment and (2) to evaluate factors infl uencing cell attachment during initial stages and subsequent steps of bacterial colonization.
Microcontact Printing of Thiols Standardized testing method needed . To quickly assess attachment of X. fastidiosa cells to a surface, we used a technique called microcontact printing ( http://education.
mrsec.wisc.edu/294.htm ). In simple wording, the technique allows the fabrication of modified surfaces on top of an inert substrate. The advantage of this technique is the creation of a surface to test surface-related phenomena, such as physical attraction, attachment, adhesion (stronger than attachment), and repulsion. Additionally, this constitutes an optimum scenario to verify the influence of chemicals that may enhance or hinder these developments. There is a clear demand from numerous food and medical industries to understand attachment to equipment surfaces. The lack of standardization and known sensitivity of methods has been pointed out [ 4 ]. The use of standard methods capable of measuring the bulk properties of cell aggreates rather than the behavior of isolated cells could provide a unique insight into understanding and controlling biofilm formation. For example, quorum sensing, a well-known phenomenon that characterizes a system of stimuli and responses correlated to population density could greatly benefit from a measurement technique that can provide insights into triggering factors. Moreover, we envision that such a technique would speed up cell surface studies because of its ability to scale up experi- mental conditions.
Why microcontact printing of thiols? . Microcontact printing of thiols (MCT) is a technique that attaches a monolayer of thiol-functionalized molecules to a silver or gold surface. The methodology was developed by George Lisensky, based on the Tollens’s Test and the well-known self-assembly of thiol monolayers (SAM) on gold surfaces [ 5 , also
http://education.mrsec.wisc.edu/294.htm ]. In other words, the surface properties of a substrate can be altered depending on the properties of the monolayer molecules. The printing can be accomplished using any pattern as long as there is a strong affinity of the printed molecules for the substrate surface.
The above website is supported by the materials science education group at the Materials Research Science and Engineering Center of the University of Wisconsin- Madison, which supports educational modules based on
doi: 10.1017/S1551929515000759
www.microscopy-today.com • 2015 September
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