Multivalency plays a major role in biological processes and particularly in the relationship between pathogenic microorganisms and their host that involves protein-glycan recognition. These interactions occur during the first steps of infection, for specific recognition between host and bacteria, but also at different stages of the immune response. The search for high-affinity ligands for studying such interactions involves the combination of carbohydrate head groups with different scaffolds and linkers generating multivalent glycocompounds with controlled spatial and topology parameters. By interfering with pathogen adhesion, such glycocompounds including glycopolymers, glycoclusters, glycodendrimers and glyconanoparticles have the potential to improve or replace antibiotic treatments that are now subverted by resistance. Multivalent glycoconjugates have also been used for stimulating the innate and adaptive immune systems, for example with carbohydrate-based vaccines. Bacteria present on their surfaces natural multivalent glycoconjugates such as lipopolysaccharides and S-layers that can also be exploited or targeted in anti-infectious strategies.
The fucose binding lectin LecB affects biofilm formation and is involved in pathogenicity of Pseudomonas aeruginosa. LecB resides in the outer membrane and can be released specifically by treatment of an outer membrane fraction with fucose suggesting that it binds to specific ligands. Here, we report that LecB binds to the outer membrane protein OprF. In an OprF-deficient P. aeruginosa mutant, LecB is no longer detectable in the membrane but instead in the culture supernatant indicating a specific interaction between LecB and OprF.
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-
The open reading frame PA1242 in the genome of Pseudomonas aeruginosa PAO1 encodes a putative protease belonging to the peptidase S8 family of subtilases. The respective enzyme termed SprP consists of an N-terminal signal peptide and a so-called S8 domain linked by a domain of unknown function (DUF). Presumably, this DUF domain defines a discrete class of Pseudomonas proteins as homologous domains can be identified almost exclusively in proteins of the genus Pseudomonas. The sprP gene was expressed in Escherichia coli and proteolytic activity was demonstrated. A P. aeruginosa ΔsprP mutant was constructed and its gene expression pattern compared to the wild-type strain by genome microarray analysis revealing altered expression levels of 218 genes. Apparently, SprP is involved in regulation of a variety of different cellular processes in P. aeruginosa including pyoverdine synthesis, denitrification, the formation of cell aggregates, and of biofilms.
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