Microscopy examination of red blood and yeast cell agglutination induced by bacterial lectins

Abstract

Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.

Systematically Modulating Aptamer Affinity and Specificity by Guanosine-to-Inosine Substitution

“…Thus, MST offers an accurate and convenient method for quantifying aptamer binding affinity. 24,25 MST analyses indicated that most inosinecontaining aptamers bound cocaine with a higher affinity than their unmodified counterparts (Table 1, Figure 2a). The Finally, the 38-GC G 25 substitution, 38-IC, showed an impressive 63-fold increase in affinity compared to the parent 38-GC aptamer.…”

Priority effects dictate community structure and alter virulence of fungal-bacterial biofilms

“…Mannose residues exist as a core component of fungal cell wall mannans and mannoproteins, and therefore are present across all morphologies of C. albicans [ 70 – 72 ]. Yeast agglutination assays are used to detect and study sugar-specificities of lectin activity such as of type I fimbriae [ 73 – 76 ]. As a proxy for C. freundii adhesion to C. albicans within our model, we used C. albicans yeast agglutination by C. freundii cell suspensions to determine that C. albicans–C.…”

A pore-forming β-trefoil lectin with specificity for the tumor-related glycosphingolipid Gb3

“…When adding SaroL-1 to rabbit red blood cells, hemolysis was observed instead of hemagglutination classically induced by multivalent lectins 40 . The hemolysis was confirmed by measuring the absorbance at 540 nm (Fig.…”