Functional expression, purification, and biochemical properties of subtilase SprP from Pseudomonas aeruginosa.

The Pseudomonas aeruginosa genome encodes a variety of different proteolytic enzymes several of which play an important role as virulence factors. Interestingly, only two of these proteases are predicted to belong to the subtilase family and we have recently studied the physiological role of the subtilase SprP. Here, we describe the functional overexpression of SprP in Escherichia coli using a novel expression and secretion system. We show that SprP is autocatalytically activated by proteolysis and exhibits optimal activity at 50°C in a pH range of 7-8. We also demonstrate a significant increase in sprP promoter activity upon growth of P. aeruginosa at 43°C indicating a role for SprP in heat shock response.

Subtilase SprP exerts pleiotropic effects in Pseudomonas aeruginosa.

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 ∆sprP 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.

Multivalent glycoconjugates as anti-pathogenic agents.

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.

Specific association of lectin LecB with the surface of Pseudomonas aeruginosa: role of outer membrane protein OprF.

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 lipase LipA (PA2862) but not LipC (PA4813) from Pseudomonas aeruginosa influences regulation of pyoverdine production and expression of the sigma factor PvdS.

A key element in iron-dependent regulation of iron metabolism and virulence-related functions for Pseudomonas aeruginosa is the sigma factor PvdS. PvdS expression itself is also influenced by iron-independent stimuli. We show that pyoverdine production and pvdS expression depend on one of the two lipases of P. aeruginosa.

Glycosylation is required for outer membrane localization of the lectin LecB in Pseudomonas aeruginosa.

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.