Probing Large Protein Adhesin Molecules on <I>Pseudomonas fluorescens</I> with Atomic Force Microscopy

Abstract

Protein adhesins are an important type of surface structure, and are components of some Gramnegative bacterial outer membranes, such as Pseudomonas fluorescens. The LapA adhesin is a major surface protein on this bacterium, which is required for irreversible adhesion and the initiation of biofilm formation. Atomic force microscopy (AFM) was used to characterize surface structures of P. fluorescens Pf0-1. The wild-type and three genetically modified strains were studied, namely a strain consisting of a single cross-over knockout mutation disrupting the lapA gene, a strain consisting of a single cross-over knockout mutation disrupting the lapB gene, (LapA is maintained in the cytoplasm and not transported to the cell surface), and a lapG deletion mutant (LapA is unable to be cleaved from the cell surface). AFM data was modeled using the Alexander-de Gennes (A-dG) relation for steric repulsion. We calculated the equilibrium layer thickness of the surface structures as well as spacing between adhesins. The wild-type strain and the lapA and lapB mutants all showed similar spacing for surface proteins. The strain lacking LapG had the smallest spacing between molecules. This suggests that the absence of the LapG protease allowed the LapA protein to accumulate, thus decreasing the overall molecular spacing of the protein on the bacterial surface compared to the wild-type strain. We found that the lapG mutant strain of P. fluorescens behaved like a classical polymer brush, in which the spacing between molecules was very small (3.3 nm), which would allow intermolecular interactions between protein units. Recent work has shown that the lapG mutant has greater adhesion and biofilm formation than the wild-type, lapA, and lapB strains, and exhibits stiffer conformation of LapA due to higher protein density and aggregation. Taken together, our results and these recent studies support the finding that LapA adhesin conformation is related to irreversible bacterial adhesion.

Atomic force microscopy-guided fractionation reveals the influence of cranberry phytochemicals on adhesion of Escherichia coli

“…A Matlab script was used, as described previously, to get the average force measurements per bacteria per treatment in nN. 28 The data were plotted and analysed using Graph Pad Prism version 6.04 for windows (GraphPad Software, La Jolla California USA). Retraction curve plots (Adhesion forces vs. Pull-off distance) depicting the trends of cranberry treatments on adhesion were created using SigmaPlot version 12.5 (Systat Software, San Jose California USA).…”

“…In the past, we have successfully demonstrated that the surface properties of uro-pathogenic bacteria can be modified by treatment with cranberry compounds. 27,28 Using a combination of microscopic techniques such as light microscopy, atomic force microscopy (AFM) and microbiology assays including growth curves, biofilm assay, we have shown that cranberry compounds are capable of altering bacterial adhesive properties and hence their ability to form biofilms. 13,27,[29][30][31] Studies performed by us and others have shown that exposure to cranberry juice can inhibit the attachment of E. coli to human uroepithelial cells, red blood cells, and resin beads with receptors that bind to surface P-fimbriae molecules.…”

“…[42][43][44] This approach has been used to detect the effect of cranberry juice on the adhesive properties of uro-pathogenic bacteria or to study the surface polymers of the bacterial strains. 28,[45][46][47][48][49] Using AFM, we have shown that CJC byproducts in urine can reduce the adhesive forces between the bacteria (E. coli and Staphylococcus aureus) and the silicone nitride tip by changing the conformation of the polymers present on the E. coli surface. 35 A similar approach is used in this study to test the anti-adhesive effect of different cranberry fractions on antibiotic resistant and pathogenic E. coli bacteria.…”