Looking Bacteria Using AFM
Quantifying Bacterial Adhesion with Force Spectroscopy
- Fig. 1: Contact mode topography image the Escherichia coli K-12 strain (csgA single-gene knockout mutant) obtained by AFM. Multiple flagella and other fimbre structures decorating the bacterial cell wall are clearly resolved.
- Fig. 2: (a) Bio-AFM image of curli protein domains on the living bacterial surface (left: before protein induction, middle: after induction, right: magnified image, reprinted from Oh. et.al. ), (b) schematic design of single molecule force spectroscopy experiments, (c) typical force-distance curves recorded using an AFM cantilever tip functionalized with RGD peptide, and (d) experimental probability density functions (equivalents of continuous histograms) of dissociation forces. Maxima represents the most probable measured dissociation force values between fibronectin and csgA monomer of different csgA mutants .
- Fig. 3: (a) Tipless cantilevers containing curli over-expressed mutant, (b) typical force-distance curves between a bacterial cell and fibronectin, and (c) histogram of detachment work upon dissociation of bacterial mutant from the FN surface .
As the bacterial outer membrane interacts with the extra-cellular environment directly, characterizing its membrane structures and binding capacities provides crucial information for understanding fundamental processes such as bacterial adhesion, and initial attachment to abiotic or biotic surfaces. Biological atomic force microscopy is the tool of choice for detailed microbial studies because it allows for studying living microbial organisms in their natural environment at the nano-scale.
It has been recently recognized that mechanical interactions play a significant role in the microbial biology on surfaces. Since the two most influential features of bacterial existence are cell-cell interaction and cell-surface interaction in natural environments, characterizing the surface structure and the binding mechanism provides crucial information for understanding fundamental processes such as bacterial adhesion, surface recognition, biofilm development, and the incipient stage of infection and disease. In addition to an understanding of the bacterial environment, many details regarding organization and physiological function need to be discovered for identifying the main molecular players involved in the interaction with abiotic and cellular surfaces in the development of biofilms.
The latest advances in atomic force microscopy (AFM) provide us with powerful tools to explore the physical and mechanical properties of bacteria in unprecedented detail. Most importantly, AFM provides ideal conditions for nano-scale imaging of microbial surfaces in their native, physiological environment and for elucidating processes occuring at the interace between micro-organisms and cells. With respect to biological imaging, bio-molecular recognition and localization of specific binding sites, bio-AFM has meanwhile developed into a sophisticated tool. It shows high potential and superior performance for force-probing the strength of receptor-ligand bonds and nano-mechanical properties of bio-molecules at the single molecular level [1-3]. Moreover, bio-AFM has revealed that nano-mechanical properties of bacteria can impose mechanics to drive adaptive behavior [4-6].
AFM Imaging of Living Bacteria
Bio-AFM, initially invented to visualize atoms, is an ideal tool for resolving filamentous structures produced by living bacterial cells and and is also apt for analyzing its biochemical and material properties in aqueous media.
The measuring principle of AFM, described in a nut shell is as follows: a sharp probe tip mounted on a micro cantilever scans horizontally line by line across a surface. Due to the interaction of the tip with the surface, the cantilever is deflected and generates a topographic image. Topographical images of surface structures reach sub-molecular resolution at the nano-scale, in real time and under physiological conditions with minimal sample preparation efforts.
We recently showed that the spatial resolution of AFM is perfectly suited for resolving structural changes of the bacterial cell surface arising from protein production . AFM topography images of different E. coli mutants clearly revealed the expression of bacterial curli CsgA fimbriae protein complexes on the outer surface of living bacterial cells (fig. 2a).
Single Molecular Force Spectroscopy (SMFS) on Bacteria
Many processes on the cellular surface rely on molecular forces that are a complex interplay of chemical, biological and physical interactions. Elucidating when and where certain interactions determine bacterial processes is key when exploring the forces involved in microbial cell adhesion. Bio-AFM opens up the possibility to measure inter- and intra-molecular forces of bio-molecules on a single molecular level with a high force detection sensitivity of a few pico Newton. In SMFS measurements, molecular interaction forces are studied in ‘so-called’ force-distance cycles. An AFM tip carrying a ligand is brought in contact with a surface that contains the respective cognate receptors, so that a receptor/ligand bond is formed. This bond is subsequently broken at a characteristic measurable dissociation force by retracting the tip from the surface. Applications of this technique include measurements of recognition forces between biotin-avidin, complementary nucleotides, cell adhesion proteins, and antibody-antigen recognition at a resolution of a few pN [1-3].
We recently probed the interaction forces between monomeric component curli protein CsgA, oligomeric curli fibers, or bacteria-carrying curli, and various fibronectin constructs (fig. 2b) . Using SMFS, we found that binding of fibronectin to CsgA is highly specific and selective. Dissociation forces between fibronectin and CsgA monomer or single bacteria were followed in force distance curves (fig. 2c). Bacterial cells containing the amyloid curli protein CsgA coupled to the extracellular matrix protein fibronectin with multiple force connections (evident from the multiple maxima in the force distributions shown in fig. 2d).
Single Microbial Cell Force Spectroscopy (SCFS)
In addition, SCFS in which AFM cantilevers are functionalized with microbial cells, opens up the possibility to quantify adhesive interactions of cells directly on a single-cell basis [4, 8]. Thus, an exciting challenge is to combine SMFS and SCFS to gain detailed insight into the molecular and cellular binding mechanisms of bacterial adhesion.
The key initial step towards infection is the binding of curli to the host’s glycoproteins such as fibronectin. We exploited SMFS to reveal essentialdetails of how curli binds to fibronectin . We additionally configured force spectroscopy studies by placing bacteria onto tip-less cantilevers and studied the interactions of single bacteria with fibronectin using SCFS measurement (fig. 3a). Different stages of curliation were mimicked by using wild type (WT), CsgA knock-out mutant (CsgA(-)), and CsgA-over-expressing mutant strains (CsgA(+)). Interaction forces between single bacteria and fibronectin coated surfaces were followed in force-distance curves (fig. 3b). Bacterial cells containing the amyloid curli proteins CsgA (CsgA(+)) bound to the extracellular matrix protein fibronectin with multiple force connections (fig. 3b). The work required to dissociate curliated bacteria from the fibronectin surface amounted to 2750 KBT, which can be taken as the upper threshold that allows bacterial cells to resist detachment from external forces (fig. 3c).
We have shown that curliated E. coli form quantized and multiple bonds of high tensile strength with fibronectin through specific RGD/CsgA connections that lead to quasi-irreversible bacterial attachment. The suchlike accomplished tight binding may allow bacteria to resist detachment from host cells induced by shear force in blood and intestinal fluids to facilitate bacterial internalization and invasion. Our insights provided by single molecule and microbial cell force spectroscopy measurements constitute the basis for unraveling novel mechanisms that govern bacteria-host cell interaction. This also offers exciting perspectives for controlling bacteria-host binding and thus opens new possibilities for alternative therapeutic strategies.
This study was funded by the Austrian Science Fund FWF projects V584-BBL, and the Austrian National Foundation for Research, Technology and Development and Research Department of the State of Upper Austria.
Yoo Jin Oh1 and Peter Hinterdorfer1
1Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria
Yoo Jin Oh, PhD
Department for Applied Experimental Biophysics
Institute of Biophysics
Johannes Kepler University Linz
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