Pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa is the leading cause of mortality among patients suffering from cystic fibrosis. Alginate-producing P. aeruginosa is uniquely associated with the environment of the cystic fibrosis-affected lung, where alginate is believed to increase resistance to both the host immune system and antibiotic therapy. Recent evidence indicates that P. aeruginosa is most resistant to antibiotics when the infecting cells are present as a biofilm, as they appear to be in the lungs of cystic fibrosis patients. Inhibition of the protective alginate barrier with nontoxic compounds targeted against alginate biosynthetic and regulatory proteins may prove useful in eradicating P. aeruginosa from this environment. Our research has dealt with elucidating the biosynthetic pathway and regulatory mechanism(s) responsible for alginate synthesis by P. aeruginosa. This review summarizes reports on the role of alginate in cystic fibrosis-associated pulmonary infections caused by P. aeruginosa and provides details about the biosynthesis and regulation of this exopolysaccharide.
The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (GIP) and glucose 6-phosphate. The Pseudomonas aeruginosa causes severe and debilitating pulmonary infections of children and young adults afflicted with cystic fibrosis (CF). P. aeruginosa isolated from the respiratory tracts of CF patients switches from a nonmucoid form to a mucoid, alginate-producing form upon progression of the disease (24). Alginate encapsulation is believed to protect the infecting bacterial cells from phagocytosis, as well as from antibiotic therapy. Alginate is a partially 0-acetylated, linear copolymer of D-mannuronate and L-guluronate linked via P-1,4-glycosidic bonds (10). Fructose 6-phosphate was identified as an alginate precursor for the P. aeruginosa biosynthetic pathway and appears to be recruited from the carbohydrate pool via the Entner-Doudoroff pathway with the participation of fructose 1,6-bisphosphate aldolase (2). Fructose 6-phosphate is converted to mannose 6-phosphate (M6P), which is subsequently converted to mannose 1-phosphate (M1P), leading to the formation of GDP-mannose and GDP-manuronic acid. A bifunctional enzyme, phosphomannose isomerase (PMI)-guanosine diphosphomannose pyrophosphorylase (GMP), is responsible for the first and third steps of the reaction (34) while phosphomannomutase (PMM) carries out the second step of the reaction (25,39 996-6415. encodes an acetylase (12, 35). Substrate specificities, functional requirements, and critical structural domains for substrate binding, catalysis, and interaction with the cofactor NAD have been characterized in detail for the two initial enzymes, PMI-GMP and GDP-mannose dehydrogenase (23, 30), while very limited information on these aspects has been obtained for the enzyme PMM.In addition to alginate, lipopolysaccharide (LPS) is another virulence factor of P. aeruginosa. Two distinct forms of LPS, the A and B bands, have been characterized. A-band LPS (D-rhamnan polysaccharide common antigen) consists mainly of a repeating trisaccharide of O-D-rhamnose, with smaller amounts of 3-O-methylrhamnose, ribose, mannose, glucose, and a 3-O-methylhexose (1). B-band LPS contains the 0 antigen (O side chain; 14, 17). A-band LPS is antigenically conserved, while B-band LPS is serologically variable. It has been shown that B-band LPS is a major virulence factor (3-5). Mutants devoid of 0 antigen were considerably more sensitive to serum components and phagocytic effects than their parent strains were. It has been recently demonstrated that the enzyme PMM is involved in LPS synthesis in P....
The exopolysaccharide alginate is a maJor virulence factor ofPseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity ofP. aeruginosa within the environment of the cystic fibrosis lung. The gene a4gC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation ofalgC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgRl. AlgRl was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgRl specifically bound with two regions ofalgC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgRl protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the aIgC promoter, which has significant homology with the RNA polymerase o-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.
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