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Chapter 2 : Methodological Approaches to Analysis of Adhesins and Adhesion

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Abstract:

The study of mechanisms of adhesion vary from simple methods providing limited information to more complex methods that are difficult to perform and analyze but provide more meaningful information. This chapter discusses some of the most important methods and emphasizes the types of knowledge gained from the use of a specific method. Bacteria that exhibit increased hydrophobicity, as determined by the contact angle technique, are more readily engulfed by phagocytes, consistent with the notion that hydrophobicity is important in adhesion. Adhesion to microspheres can be determined by light microscopy, among many other techniques (enzyme-linked immunosorbent assay (ELISA) and use of radiolabeled bacteria). Hemagglutination reactions have been responsible for the initial identification of many lectin adhesins and were important in the characterization of adhesin-saccharide specificities, through the use of carbohydrate inhibitors of the reactions. In approaching important subjects such as affinity, the chapter mentions two basic types of experiments that can be performed. The usefulness of the kinetic approach is extended when experimental variables are manipulated. Methods have been developed to easily differentiate between extracellularly bound and internalized bacteria. One of simplest tests to determine whether an adhesin is expressed in vivo during a natural infection is to assay for antibodies against the adhesin in patient sera. Another relatively easy assay is to screen isolates from an infection for mRNA for an adhesin by reverse transcription-PCR.

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2

Key Concept Ranking

Type 1 Fimbriae
0.46676776
Enzyme-Linked Immunosorbent Assay
0.464433
Scanning Electron Microscopy
0.4395369
Atomic Force Microscopy
0.43656707
0.46676776
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Image of FIGURE 2.1
FIGURE 2.1

(A) Diagrammatic representation of the interaction of hydrophobic and of hydrophilic bacteria with hexadecane droplets. The bacterial suspension is overlaid with hexadecane, usually at a 1:10 ratio of hexadecane to aqueous solution (e.g., 0.1 ml of hexadecane plus 1.0 ml of bacterial suspension). After vigorous vortexing for 1 min, the hexadecane breaks down within the bacterial suspension into droplets of various sizes. Bacteria will attach in numbers that are relative to the degree of surface hydrophobicity. After the mixed phases are allowed to separate for a few minutes, the hexadecane droplets will rise to the top, carrying the bound hydrophobic bacteria. Hydrophilic bacteria will remain in relatively greater numbers in the lower (aqueous) phase because they do not bind to the hydrocarbon. (B) Spectrophometric determination of the optical density of the aqueous phase after allowing phase separation to occur will allow the determination of the relative hydrophobicity of bacterial populations. The less turbid aqueous phase contains the bacteria with the most hydrophobic surfaces. This assay can be used to fractionate hydrophobic and hydrophilic bacteria in a mixed population. (C and D) Light micrographs (low [C] and high [D] magnifications) of bacteria bound to hexadecane droplets.

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Image of FIGURE 2.2
FIGURE 2.2

Schematic diagram showing the effects of exposure of hydrophobin and protein adhesins (or masking by capsule) on the binding of streptococci to hexadecane droplets. (A) The hyaluronate capsule masks the LTA hydrophobin, and as a result the bacteria cannot adhere to the hexadecane droplets. (B) The hydrophobin is exposed in naturally unencapsulated streptococci (e.g., stationary phase), and so the bacteria are able to bind to hexadecane droplets. The same would be true for bacteria treated with hyaluronidase to remove the capsule. (C) Removal of the hydrophobin by enzymatic digestion of the bacteria makes the bacteria hydrophilic and, therefore, unable to bind to hexadecane. See the text and reference 93 for more detail.

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Image of FIGURE 2.3
FIGURE 2.3

Flow cytometric analysis of adhesion of fluorescein-labeled to HEp-2 tissue culture cells. The flow chamber is gated to detect epithelial cell-sized particles as a function of the level of fluorescence. The figure shows light scattering of epithelial cells alone (A) and epithelial cells 10 min after being mixed with the bacteria (B). Data were extracted from fluorescence analysis of epithelial cells and depicted as dot plots of forward scatter versus side scatter. A shift of the peak of fluorescence intensity to the right indicates increase in fluorescence intensity of the epithelial cells caused by adhesion of bacteria to the cells. The appearance of a shoulder on the right of the histogram indicates that a subpopulation of cells binds an increased number of bacteria. (Reprinted from reference with permission from the publisher.)

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Image of FIGURE 2.4
FIGURE 2.4

Identification of receptors by using blots of isolated membrane glycoproteins. Asymmetric unit membrane plaques were purified from bladder uroepithelial cells and separated by SDS-PAGE. These membrane plaques are enriched for uroplakin (UP) proteins Ia, Ib, II, and III. (A) Electrophoretic pattern of bovine urothelial plaque proteins stained with silver nitrate (lane 2). Lane 1 contains molecular weight markers. (B) Identification of uroplakin proteins by immunoblotting with antibodies (Ab) against synthetic peptides mimicking proteins Ia (lane 1), Ib (lane 2), II (lane 3), and III (lane 4). (C) Binding of radiolabeled to uroplakins. After blotting SDS-PAGE-separated uroplakins to nitrocellulose, membranes were treated as follows: those in lane 1 were incubated with J96, which expresses both type 1 and P fimbriae; those in lane 2 were incubated with SH48, which expresses only type 1 fimbriae; those in lane 3 were incubated with HU849, which expresses only P fimbriae; those in lane 4 were incubated with P6678-54, which expresses neither type 1 nor P fimbriae. (D) Uroplakins were deglycosylated by treatment with control buffer (lanes 1, 4, and 7) or with endo H to remove high-mannose-type sugars (lanes 2, 5, and 8) or endo F (lanes 3, 6, and 9). Following deglycosylation, uroplakins were separated by SDS-PAGE and blotted to nitrocellulose, and the membranes were probed by using uroplakin-specific antibodies. Note the removal of ~3-kDa equivalents of the high-mannose-type sugars from uroplakin Ia and Ib by endo H and the removal of ~20-kDa equivalents of sugars, most probably the complex type, from uroplakin III. (E) Binding of radiolabeled to deglycosylated uroplakins. Before SDS-PAGE and blotting, uroplakins had been treated with control buffer (lanes 1 and 4), endo H (lanes 2 and 5), or endo F (lanes 3 and 6). The membranes were incubated with radiolabeled J96 (lanes 1 to 3), or SH48 (lanes 4 to 6). The binding of type 1-fimbriated to uroplakin Ia and Ib was abolished by endo H and endo F treatment, suggesting the involvement of a high-mannose-type saccharide in the binding. (Reprinted from reference with permission from the publisher.)

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Image of FIGURE 2.5
FIGURE 2.5

Identification of fimbrial glycolipid receptors by TLC. Purified glycolipids were separated by TLC and stained with orcinol (A and C), or probed with F1C fimbriae purified from and immunostained with antifimbrial antibodies (B and D). GlcCer, glucosylceramide; SFT, sulfatide; GgOCer, asialo-GM ganglioside; GM1, sialylated GM ganglioside; GalCer2, galactosylceramide; LacCer, lactosylceramide; nLcCer, paragloboside; GgOCer, asialo-GM ganglioside; GbCer, globotriaosylceramide; GbCer, globotetraosylceramide. Using this assay and other binding assays, the disaccharide sequence GalNAc(β1→4)GalB of asialo-GM and asialo-GM could be identified as the high-affinity binding epitope for F1C fimbriae of uropathogenic (Reprinted from reference with permission from the publisher.)

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Image of FIGURE 2.6
FIGURE 2.6

Scanning electron micrographs of human small intestinal mucosa infected with enteropathogenic (A) Large areas of mucosal surface can be observed easily at low magnification. (B) Finer details of adhesion can be observed at higher magnification. Here, the bacteria can be seen to adhere intimately, dramatically altering the brush border of the enterocytes. (Reprinted from reference with permission from the publisher.)

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Image of FIGURE 2.7
FIGURE 2.7

Kinetic method for evaluating bacterial adhesion to a substratum. The model depicts two steps of adhesion, each with separate association and dissociation constants. One represents weak, reversible adhesion, and the other represents firm adhesion. See the text for additional details.

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
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Tables

Generic image for table
TABLE 2.1

Types of target cells and particles commonly used in agglutination reactions with bacteria

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.2

Methods commonly used to separate nonadherent bacteria from bacteria adherent to animate surfaces

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.3

Methods commonly used to separate nonadherent bacteria from bacteria adherent to inanimate surfaces

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.4

Methods commonly used to quantify adherent bacteria

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.5

Methods commonly used to distinguish between extracellularly bound and internalized bacteria

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.6

Suggested progression of steps to be taken for the identification of new bacterial adhesins

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.7

Suggested progression of steps to be taken for the identification of new adhesin receptors

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2
Generic image for table
TABLE 2.8

Methods used to assess the role of adhesion in infection

Citation: Ofek I, Hasty D, Doyle R. 2003. Methodological Approaches to Analysis of Adhesins and Adhesion, p 19-42. In Bacterial Adhesion to Animal Cells and Tissues. ASM Press, Washington, DC. doi: 10.1128/9781555817800.ch2

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