J B Goldberg

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Pseudomonas aeruginosa AlgB, which modulates the expression of alginate, is a member of the NtrC subclass of prokaryotic regulators

The Pseudomonas aeruginosa exopolysaccharide alginate is an important virulence factor in chronic pulmonary infections of cystic fibrosis patients. We determined the nucleotide sequence of the gene, algB, which regulates the level of exopolysaccharide produced by mucoid P. aeruginosa. The predicted amino acid sequence of AlgB revealed a high degree of similarity to the regulatory proteins in the NtrC subclass of ‘two‐component regulatory systems’. AlgB expression in Escherichia coli minicells showed a molecular weight of ˜ 50 000 Da, comparable to that of the inferred amino acid sequence (49 318 Da). We show that algB is transcriptionally active in mucoid strains of P. aeruginosa and regulates the expression of the alginate biosynthetic gene, algD, thereby resulting in increased expression of alginate in mucoid P. aeruginosa.

The rfb locus from Pseudomonas aeruginosa strain PA103 promotes the expression of O antigen by both LPS-rough and LPS-smooth isolates from cystic fibrosis patients

Summary

Cloning and characterization of the gene (rfc) encoding O-antigen polymerase of Pseudomonas aeruginosa PAO1

The lipopolysaccharide (LPS) O-antigen polymerase is the product of the rfc gene. Loss of O-antigen polymerase activity due to mutation in rfc gives rise to a characteristic LPS phenotype known as core-plus-one or semi-rough, wherein the LPS core is capped with a single oligosaccharide unit. Pseudomonas aeruginosa (Pa) AK1401, a derivative of strain PAO1 (serogroup O5), expresses a semi-rough LPS; this mutant phenotype was complemented by a 2.2-kb NsiI-SacI fragment of Pa PAO1 DNA. Sequence analysis of this fragment revealed a 1317-bp open reading frame (ORF) potentially encoding a 438-amino-acid (aa) protein of 48,849 Da. This DNA sequence and the inferred aa sequence contain many of the features of other O-antigen polymerases, including an aberrantly low G + C content (particularly apparent in the high-G + C background of Pa), an unusual codon usage pattern, and a hydrophobicity profile indicative of a membrane protein. A 345-bp fragment internal to the ORF hybridized to genomic DNA from two of ten Pa serogroup strains examined by Southern blot; these two strains express O antigens structurally related to that of strain PAO1.