Subject(s)
Community Health Centers/organization & administration , Family Practice/organization & administration , Job Satisfaction , Personnel Turnover , Community Health Centers/economics , Family Practice/economics , Humans , Medically Uninsured , Ohio , Physician-Patient Relations , Professional AutonomyABSTRACT
A series of 5-(2- or 3-thienyl)tryptamine derivatives (9) has been synthesized and shown to be potent and selective 5-HT1D versus 5-HT1B receptor agonists and, therefore, potential treatments for migraine.
Subject(s)
Migraine Disorders/drug therapy , Receptors, Serotonin/drug effects , Serotonin Antagonists/chemical synthesis , Tryptamines/chemical synthesis , Cloning, Molecular , Humans , Indicators and Reagents , Receptor, Serotonin, 5-HT1B , Receptor, Serotonin, 5-HT1D , Serotonin Antagonists/pharmacology , Structure-Activity Relationship , Tryptamines/pharmacologyABSTRACT
In Klebsiella pneumoniae, the ability to synthesize large amounts of capsular polysaccharide is an important correlate of virulence. We report the cloning of rcsA from K. pneumoniae serotype O1:K20 and demonstrate that rcsA is involved in the expression of the K antigen capsule. We have determined the nucleotide sequence for the rcsA gene from K. pneumoniae K20 and shown it to be identical to the sequence reported previously for rcsA from strain K21 (Allen et al., J. Gen. Microbiol. 133:331-340, 1987). Southern hybridization results indicate that this gene is widely distributed among different Klebsiella K serotypes. When cloned into Escherichia coli K-12, the K. pneumoniae rcsA gene caused a mucoid phenotype, resulting from the activation of colanic acid synthesis. Activation of colanic acid synthesis was not dependent on growth at low temperatures (less than or equal to 30 degrees C). The K. pneumoniae rcsA gene complemented E. coli K-12 rcsA mutations but could not complement defects in rcsB, suggesting that RcsA may be functionally homologous in these bacteria. The cloned rcsA gene also complemented a defect in nonmucoid strain K20 derivatives that normally produced only trace amounts of K20 antigen and were unable to assemble a wild-type capsular structure. Mutants that were K20-deficient were not complemented. The K antigen capsule of K. pneumoniae therefore joins a growing list of polysaccharide-synthetic systems in which "RcsA-like" proteins are involved.
Subject(s)
Antigens, Bacterial/genetics , Antigens, Surface/genetics , Genes, Bacterial , Genes, Regulator , Klebsiella pneumoniae/genetics , Polysaccharides, Bacterial/genetics , Base Sequence , Chromosome Mapping , Cloning, Molecular , Genetic Complementation Test , Klebsiella pneumoniae/immunology , Klebsiella pneumoniae/ultrastructure , Polysaccharides/biosynthesis , Polysaccharides, Bacterial/biosynthesisABSTRACT
The lipopolysaccharide (LPS) O-antigen side chains of Klebsiella serotype O1 have been studied by using mutants selected by resistance to a Klebsiella bacteriophage designated O1-A. Two classes of LPS mutants were identified. The major group (90%) synthesized rough LPS. The remaining 10% of the mutants produced a novel LPS profile that lacked the highest-molecular-weight O-substituted molecules (HMW-LPS) but still produced lower-molecular-weight O-substituted species (LMW-LPS). By using antisera raised against mutant Klebsiella strains and antiserum specific for Pasteurella haemolytica serotype 4, it was demonstrated that HMW-LPS and LMW-LPS contain shared epitopes. HMW-LPS also contained an epitope absent in LMW-LPS. This unique epitope was recognized by a monoclonal antibody (O1-52.6) and appears to be responsible for the serological cross-reaction between the O antigens of Klebsiella O1 and Escherichia coli O19. This HMW-LPS epitope was present in eight other Klebsiella O1 isolates which were examined. Electron microscopy demonstrated that HMW-LPS excluded overlying capsular polysaccharide for a distance of 25 to 40 nm. The distance was reduced to 10 to 18 nm in strains which synthesized only LMW-LPS and to zero in rough LPS strains. The HMW-LPS of Klebsiella O1 was shown to be an important virulence determinant, since this molecule was responsible for the resistance of the bacterium to nonspecific, complement-mediated serum killing.
Subject(s)
Antigens, Bacterial/immunology , Klebsiella pneumoniae/immunology , Lipopolysaccharides/immunology , Animals , Bacteriophages/growth & development , Blood Bactericidal Activity , Epitopes , In Vitro Techniques , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/ultrastructure , Microscopy, Electron , Molecular Weight , O Antigens , Rabbits , SerotypingABSTRACT
Coliphage K30 lysates contain free and phage-associated forms of a bacteriophage-encoded capsule depolymerase (glycanase) enzyme, active against the serotype K30 capsular polysaccharide of Escherichia coli. The free glycanase has been purified to apparent homogeneity. The molecular weight of the enzyme was estimated at 450,000, and when heated in SDS at 100 degrees C, the enzyme dissociated into two subunits of 90,000 and 52,000. The glycanase enzyme was used as a reagent to reversibly degrade the capsular layers on cells of Escherichia coli O9:K30 and Klebsiella O1:K20. This treatment rendered these bacteria sensitive to their respective lipopolysaccharide-specific bacteriophages, coliphage O9-1 and Klebsiella phage O1-3. This novel approach facilitated isolation of lipopolysaccharide O antigen side chain deficient mutants which retained the ability to synthesize the capsule. The response of defined mutants, O+:K-, O-:K+, and O-:K-, to exposure to nonimmune rabbit serum was measured. Results showed that the primary barrier against complement-mediated serum killing in both Escherichia coli O9:K30 and Klebsiella O1:K20 was the O antigen side chains of the lipopolysaccharide molecules. In both strains, the capsule played no role in the determination of serum resistance.
