Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation

Abstract

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative B max values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K d and relative B max , which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligandreceptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire

“…For this purpose, we turned our attention to the surface glycans that had already been described in Acanthamoeba: glucose, mannose, GalNAc, and GlcNAc (10). APMV particles were incubated in solutions with each of these glycans at a concentration of 10, 25, 50, 75, 100, or 125 g/ml, followed by incubation with A. castellanii cells.…”

“…To evaluate the role of glycans in viral adhesion to distinct organisms, 10 6 TCID 50 of viruses (APMV, LAPMV, DAPMV, and mimivirus M4) treated with various concentrations of different carbohydrates (glucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, chitin, and peptidoglycan) were incubated with the same amount of microorganism cells and mosquito legs using the same conditions described above. The results were plotted in a column graph, and the statistical significance was given by a two-tailed Student's t test, performed using GraphPad Prism software.…”

“…It is known that for phagocytosis to occur, the microbial structure and cellular adhesion factors, which are often surface glycans, must interact (9). On the surface of Acanthamoeba, glucose, mannose, N-acetylgalactosamine (GalNAc), and Nacetylglucosamine (GlcNAc) have already been identified to be cellular adhesion factors (10). These carbohydrates serve as the building blocks for several natural polymers, such as peptidoglycan and chitin, both of which are largely distributed in the microbial world as the structural components of bacteria, fungi, and arthropods, among other organisms.…”

Functional roles of glycogene and N‐glycan in multidrug resistance of human breast cancer cells

“…A major population of N-glycans detected in MCF-7 and MCF/ADR cells corresponded to high-mannose structures, and some high mannoses in particular peak 2, 8, 17 were dramatically increased in drug-resistant MCF/ADR cell line. Moreover, MCF/ADR cells showed a significant increase of fucosylated and sialylated oligosaccharides (peak 3,5,10,12,16,20,22). Therefore, monitoring of the N-glycan profile would be an important step in the prevention of tumor MDR and would increase our understanding of resistance mechanisms.…”

Exploring the Unique N-Glycome of the Opportunistic Human Pathogen Acanthamoeba

“…Although there are various earlier reports on a partial characterization of lipophosphonoglycan (10) and the presence of a N-acetylglucosaminyl-1-phosphotransferase (11) as well as various radiolabeling (12), lectin binding (12)(13)(14)(15) or monosaccharide composition studies (16), our present data are the first regarding the actual structures of glycosylated macromolecules of this genus. As it has been shown that many protozoal parasites synthesize highly unique N-glycans (17), it can be hypothesized that the investigation of the N-glycosylation of parasitic and nonparasitic Acanthamoeba strains may yield insights into virulence factors.…”