Expression of the Klebsiella pneumoniae alginate lyase gene in Pseudomonas aeruginosa--effect on alginate structure.

Mucoid or alginate-producing strains of the opportunistic pathogen Pseudomoms aeruginosa frequently colonise the respiratory tract of patients with cystic fibrosis (CF). The virulence factor alginate protects the bacterium from the host immune system and aggnssive antibiotic therapies, thus causing an intractable chronic pulmonary infection which is recognised as a major cause of morbidity and mortality amongst these patients. Alginates are linear exopolysaccarides comprising (1 +)-linked &D-mannuronate (M) and its CS-epimer a-L-guluronate (G). These residues are arranged in block structures which may be [poly-mannuronate (poly-MM) or poly-guluronate (poly-GG)] or random heteropolymeric sequences (poly-MG). Alginate produced by P.aeruginosa lacks poly-GG blocks and the mannuronate residues may be either monoor di-0-acetylated [l]. At present the clinical management of mucoid P. aeruginosu infections in the CF lung requires the administration of potent antibiotics and a complex regime of physiotherapy. These measures however conml the infection only transiently and the micro-organism is never totally eradicated. One suggested therapy has been the use of alginate lyases to degrade and aid the dispersal of alginate allowing the host immune system and conventional treatments an opportunity to clear the pulmonary infection [l]. In an in vitro model addition of an alginate lyase has been shown to enhance the uptake of mucoid P. aeruginosa by macrophages [2]. Alginatc lyases have been isolated from many sources, and are generally endo-acting enzymes that catalyse the blimination of the 4-0-linked glycosidic bond of alginates with the formation of unsaturated uronic-acid-containing oligosaccharides [3]. They all have an absolute specificity for either mannuronate or guluronate residues at the non-reducing end of the bond to be cleaved [4]. Guluronate-sptcific lyases are potentially better therapeutic agents in the treatment of mucoid P. aeruginosu infections as bacterial alginates contain 0-acetylated mannuronate residues and are therefore more resistant to degradation [2]. The Klebsiella pneumonia guluronate-specific alginate lyase gene has been sub-cloned into pLAFX3 to produce the construct pALY8 [5 , 61. In this study, 'H-nmr has been used to investigate the effects of the overexpression of the K.pneumoniae alginate lyase on alginate structure in the mucoid P. aeruginosa strain 2192. Experimental procedures have been described previously [7]. Results (Table 1) indicate that the crude and purified extracellular material produced by both pLAFR3and pALY8-transconjugants contained carbohydrate, uronic acid and minor quantities of protein.