Glycosylation Is Required for Outer Membrane Localization of the Lectin LecB inPseudomonas aeruginosa

Abstract

The fucose-/mannose-specific lectin LecB from Pseudomonas aeruginosa is transported to the outer membrane; however, the mechanism used is not known so far. Here, we report that LecB is present in the periplasm of P. aeruginosa in two variants of different sizes. Both were functional and could be purified by their affinity to mannose. The difference in size was shown by a specific enzyme assay to be a result of N glycosylation, and inactivation of the glycosylation sites was shown by site-directed mutagenesis. Furthermore, we demonstrate that this glycosylation is required for the transport of LecB.Lectins are proteins of nonimmune origin that recognize and bind to specific carbohydrate structural epitopes without modifying them. This group of carbohydrate proteins function as central mediators of information transfer in biological systems and perform their duties by interacting with glycoproteins, glycolipids, and oligosaccharides (34). Lectins exist in a wide range of organisms, including viruses, bacteria, plants, and animals, and are believed to play a general and important role in cell-cell interactions (9). Many bacteria have an arsenal of different lectins for targeting glycosylated proteins of the host (21). One example, the lectin FimH at the top of type 1 pili from the uropathogenic Escherichia coli, recognizes terminally located D-mannose moieties on cell-bound glycoproteins to mediate adhesion between the bacterium and the urothelium (4, 20). Lectins are also of interest for medical and pharmaceutical applications, as exemplified by the galactoside-specific mistletoe lectin, which is widely used as a drug to support anticancer therapy (5).Pseudomonas aeruginosa, an opportunistic pathogen associated with chronic airway infections, synthesizes two lectins, LecA and LecB (formerly also named PA-IL and PA-IIL) (11). Strains of P. aeruginosa which produce high levels of these virulence factors exhibit an increased virulence potential (12). Both lectins play a prominent role in human infection, since it was demonstrated that P. aeruginosa-induced otitis externa diffusa (46), as well as P. aeruginosa in respiratory tract infections (56) and cystic fibrosis patients (16), could successfully be treated by the application of a solution containing specific sugars. The sugar solutions presumably prevented lectin-mediated bacterial adhesion to the corresponding host cells. The expression of lectin genes in P. aeruginosa is coordinately regulated with certain other virulence factors and controlled via the quorum-sensing cascade and by the alternative sigma fac-

Structural Determinants for Activity and Specificity of the Bacterial Toxin LlpA

“…The translocation of the outer membrane-associated mannose/fucose-specific lectin LecB of P. aeruginosa , that also lacks such signal sequence [50], is dependent on its glycosylation [51]. Contrary to LlpA that is exported to the culture supernatant to exert its antagonistic activity, LecB remains associated with the cell envelope through interaction with the major outer membrane protein OprF [52], in line with its role in biofilm formation.…”

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor

“…5E), which suggests its association with the membrane lipids. To investigate the biological significance of protein glycosylation in subcellular localization of proteins (45) and the appearance of LprI on the cell wall in addition to its membrane association, we checked whether glycosylation has any role in surface association of LprI in M. tuberculosis. Interestingly, in the modeled structure of LprI, glycosylation sites Thr-24, Thr-28, and Thr-117 are arranged on the protein surface in the same line, and among these, the Thr-24 and Thr-28 sites are close to the protein anchorage site (Cys-16) that might contribute to its surface exposure (Fig.…”

Glycodendrimers and Their Derivatives as Potential Therapeutic Agents

“…Good results have been obtained with calixarenes, β-peptoid (4), porphyrins and resorcinarenes. [11] Among these molecules, 1,3-alternate conformer calixarene showed the greatest efficiency and increase in affinity. Chelate binding mode with two α-D-galactose residues that interact with two adjacent binding sites in a single tetramer LecA, can be confirmed by the observation of well-defined nanoscale fibers complex lectin-glycoclaster by studying the atomic force microscopy (AFM).…”