Biomedical subjects

C Whitfield

Publications and source records attributed to C Whitfield.

At least 73 records · Page 4Linked to original sources

PubMed1991

The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography.

A high-performance liquid chromatography (HPLC) method with pulsed-amperometric detection (PAD) was developed for the compositional analysis of the acidic, neutral, and basic monosaccharides recovered from the acid hydrolysis of bacterial cell wall polysaccharides. This HPLC-PAD method involved the chromatography of the acid hydrolysis products on a CarboPac PA-1 anion-exchange column of pellicular resin, with PAD detection following postcolumn addition of alkali. Complete resolution of a mixture of 19 monosaccharides, comprising 9 neutral, 3 basic, and 7 acidic sugars, frequently found in bacterial polysaccharides was achieved within 60 min by the system. The presence of amino acids in the mixture was shown not to affect the analysis. This protocol was applied to the compositional analysis of 2 extracellular polysaccharides produced by Escherichia coli, colanic acid, and K30 antigen, which share constituent monosaccharides. The overproduction of extracellular polysaccharide in E. coli CWG56 was shown to be a consequence of deregulation of K30 biosynthesis and not of coexpression of an additional polymer.

Chromatography, High Pressure Liquid↗

PubMed1991

The rcsA gene of Klebsiella pneumoniae O1:K20 is involved in expression of the serotype-specific K (capsular) antigen.

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.

Antigens, Bacterial↗

PubMed1991

Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.

The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D-Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher-molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster.

Antigens, Bacterial↗

PubMed1990

Monoclonal antibodies against serotype specific and conserved epitopes in morphotype E Escherichia coli flagellins.

Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype strains belonging to morphotype E. Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45. Several mAbs which recognised conserved epitopes were also examined. mAb 7-56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filament surface. Similarly, mAb 1-5.1 recognised an internal epitope shared only by serotypes H1 and H12. Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments.

Antibodies, Monoclonal↗

PubMed1989

Mutants of Escherichia coli O9:K30 with altered synthesis and expression of the capsular K30 antigen.

Escherichia coli K30 produces a thermostable group I capsular polysaccharide. Two classes of mutants were isolated with defects in the synthesis or expression of capsule. The most common mutant phenotype was acapsular (K-), with no K-antigen synthesized. A second class of mutants, termed Ki or intermediate forms, produced colonies which were indistinguishable from those of acapsular forms yet K-antigenicity was expressed. Previous studies had demonstrated that E. coli strains that produce K30 antigen synthesize a lipopolysaccharide (LPS) fraction that is recognised by monoclonal antibodies against the K30 antigen. Synthesis of this LPS fraction was not affected in Ki forms. The results of morphological examination, LPS analysis and phage sensitivity studies are consistent with the interpretation that the defect in Ki strains results from an inability to polymerize the K30 antigen. Using plasmid pULB113 (RP4::mini-Mu), mutations resulting in both K- and Ki phenotypes were localized near the his region of the chromosome.

Antigens, Bacterial↗

PubMed1989

A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.

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.

Animals↗

PubMed1989

Use of a bacteriophage-encoded glycanase enzyme in the generation of lipopolysaccharide O side chain deficient mutants of Escherichia coli O9:K30 and Klebsiella O1:K20: role of O and K antigens in resistance to complement-mediated serum killing.

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.

Antigens, Bacterial↗

PubMed1988

Serotype-specific monoclonal antibodies against the H12 flagellar antigen of Escherichia coli.

The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament. Re-examination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology. Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the Mr of individual flagellins. There was no apparent correlation between these two features. Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella. In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different O:K antigen combinations. The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains. The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers. The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament. The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure.

Antibodies, Bacterial↗

PubMed1988

Bacterial extracellular polysaccharides.

The synthesis of extracellular polysaccharides has been recognized in certain bacterial cultures since the 1880s. It is now apparent that a wide range of bacteria produce these polymers and an equally wide range of chemical structures are possible. Their surface location, together with the range of available monosaccharide combinations, noncarbohydrate substituents, and linkage types, make extracellular polysaccharides excellent agents of diversity. As a result, much effort has been directed towards elucidating their structure in pathogenic bacteria and in enteric organisms in particular. Commercial applications of microbial polysaccharides have now broadened the scope of structural information. Most recently, technological advances in molecular biology have created the possibility of manipulating desired polymer characteristics, and with these advances, our knowledge of the mechanisms of synthesis and regulation of cell surface polysaccharides has improved. Ultimately more information regarding the function of extracellular polysaccharides in natural environments may result.

Bacteria↗

PubMed1988

Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20, in Escherichia coli K12.

Escherichia coli serotype O9:K(A)30 and Klebsiella O1:K20 produce thermostable capsular polysaccharides or K antigens, which are chemically and serologically indistinguishable. Plasmid pULB113 (RP4::mini-Mu) has been used to mediate chromosomal transfer from E. coli O9:K30 and Klebsiella O1:K20 to a multiply marked, unencapsulated, E. coli K12 recipient. Analysis of the cell surface antigens of the transconjugants confirmed previous reports that the genetic determinants for the E. coli K(A) antigens are located near the his and rfb (O antigen) loci on the E. coli linkage map. The Klebsiella K20 capsule genes were also found to be in close proximity to the his and rfb loci. Electron microscopy revealed significant differences in the structural organization of capsular polysaccharides in these two microorganisms and the morphological differences were also readily apparent in transconjugants expressing the respective K antigens. These results are consistent with the interpretation that at least some of the organizational properties of capsular polysaccharides may be genetically determined, rather than being a function of the outer membrane to which the capsular polysaccharides are ultimately attached.

Antigens, Bacterial↗

PubMed1988

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface.

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.

Animals↗

PubMed1986

Association of alginate from Pseudomonas aeruginosa with two forms of heparin-binding lectin isolated from rat lung.

An endogenous heparin-binding lectin activity isolated from rat lung was separated into two distinct isolectin forms which showed subtle changes in carbohydrate specificity. The two lectin forms displayed different specificities toward alginic acid-purified cystic fibrosis isolates of Pseudomonas aeruginosa when assayed by inhibition of both hemagglutination and [3H]heparin binding. This ability of isolectin forms to show higher affinity toward alginic acid from certain P. aeruginosa strains may suggest that there is a selective mechanism in the colonization of patients with cystic fibrosis.

Alginates↗

PubMed1986

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

Animals↗

PubMed1985

Antibiotic resistance of Pseudomonas aeruginosa colonizing a urinary catheter in vitro.

A modified Robbins Device was used to establish coherent biofilms of Pseudomonas aeruginosa on the surface of catheter material in an artificial urine milieu and the ability of an antibiotic to penetrate the biofilm and kill the enclosed bacteria was assessed. The Pseudomonas aeruginosa strain used had been isolated from a patient with urinary tract infection. Although planktonic (floating) cells of the Pseudomonas aeruginosa strain were inhibited by less than 1 mg/l of tobramycin and killed by 50 mg/l, contact with 1,000 mg/l of tobramycin for 12 h failed to kill all the sessile (adherent) bacteria in the biofilms on the surface of the catheter material. Surviving sessile bacteria recovered directly from the exposure to 1,000 mg/l of tobramycin were inhibited by 0.4 mg/l of this agent when tested as dispersed planktonic cells by standard MIC methods. It is suggested that growth within thick adherent biofilms confers upon cells of Pseudomonas aeruginosa a large measure of resistance to aminoglycosides and other antibiotics that may help to explain the frequent failure of antibiotic chemotherapy in catheter-associated urinary tract infections.

Anti-Bacterial Agents↗

PubMed1985

Membrane proteins correlated with expression of the polysialic acid capsule in Escherichia coli K1.

Growth of Escherichia coli K1 strains at 15 degrees C results in a defect in the synthesis or assembly of the K1 polysialic acid capsule. Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C, and activation requires protein synthesis (Whitfield et al., J. Bacteriol. 159:321-328, 1984). Using this temperature-induced defect, we determined the molecular weights and locations of membrane proteins correlated with the expression of K1 (polysialosyl) capsular antigen. Pulse-labeling experiments demonstrated the presence of 11 proteins whose synthesis was correlated with capsule appearance at the cell surface. Using the differential solubility of inner and outer membranes in the detergent Sarkosyl, we localized five of the proteins in the outer membrane and four in the inner membrane. The subcellular location of two of the proteins was not determined. Five proteins appeared in the membrane simultaneously with the initial expression of the K1 capsule at the cell surface. One of these proteins, a 40,000-dalton protein localized in the outer membrane, was identified as porin protein K, which previously has been shown to be present in the outer membrane of encapsulated E. coli. The possible role of these proteins in the synthesis of the polysialosyl capsule is discussed.

Bacterial Outer Membrane Proteins↗

PubMed1984

Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1. Role of a low-density vesicle fraction in activation of the endogenous synthesis of sialyl polymers.

Escherichia coli K1 synthesizes a polysialic acid capsule when grown at 37 but not 15 degrees C. The derangement in sialyl polymer synthesis appears to result from the inability of 15 degrees C membranes to synthesize or assemble a functional endogenous acceptor (Troy, F.A., and McCloskey, M.A. (1979) J. Biol. Chem. 254, 7377-7387). Membranes from cells grown at 15 degrees C spontaneously gained the ability to synthesize sialyl polymer after incubation at 33 degrees C for 2-4 h. The incubation-dependent activation of the endogenous synthesis of sialyl polymer in 15 degrees C membranes possessed two unusual features. First, the sialyltransferase was localized in a low density vesicle fraction (LDV; rho = 1.11 g/cm3). Second, this fraction catalyzed protein synthesis, and protein synthesis was required for activation. A study of the LDV fraction showed: 1) their light density resulted from a 5- to 8-fold enrichment in lipid phosphate to protein ratio and their sialyltransferase activity was enriched 40-fold compared with unfractionated total membranes; 2) they contained proteins characteristic of inner and outer membranes including leader peptidase and lipoprotein; 3) they constituted 8% of the mass of unfractionated total membranes yet contained all of the endogenous sialyltransferase activity in 15 degrees C membranes. In contrast, LDV from 37 degrees C grown cells accounted for 4.8% of the membrane mass and only 12.5% of the endogenous sialyltransferase activity; 4) they were multilamellar and averaged 0.7 mu in diameter. Based on these results, we believe the LDV fraction is of physiological importance in sialyl polymer synthesis. Growth at 15 degrees C allowed identification and study of the LDV fraction possibly because of the altered thermotropic properties of the membrane phospholipids that occur when E. coli is grown at low temperature.

Amino Acids↗

PubMed1984

Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1. Activation of sialyl polymer synthesis in inactivate sialyltransferase complexes requires protein synthesis.

The polysialosyl capsule in Escherichia coli K1 is not synthesized when cells are grown at 15 degrees C. Membranous sialyltransferase complexes isolated from cells grown at 15 degrees C spontaneously gain the ability to synthesize sialyl polymers when incubated at 33 degrees C for 2-4 h. Activation of the endogenous synthesis of polysialic acid is localized in a low-density vesicle fraction (Whitfield, C., Adams, D.A., and Troy, F.A. (1984) J. Biol. Chem. 259, 12769-12775). The low density vesicles catalyzed protein synthesis, and spontaneous activation required protein synthesis. Immune blotting confirmed the presence of protein synthesis initiation factor, IF2, and ribosomal protein S6, in the low-density vesicle fraction. Temperature-upshift experiments identified four membrane proteins whose synthesis was temporally correlated with in vitro activation. These proteins have apparent molecular masses of 40, 21, 17.5, and 16 kDA. Although the function of these proteins is not known, they may relate to a sialyltransferase activity required to initiate sialyl polymer assembly, to an endogenous acceptor, or to a porin that may facilitate channeling of the polysialosyl chains through the outer membrane.

2,4-Dinitrophenol↗

PubMed1984

Protein synthesis is required for in vivo activation of polysialic acid capsule synthesis in Escherichia coli K1.

The kinetics of in vivo expression of the polysialosyl (K1) capsular antigen in Escherichia coli has been studied. Growth of E. coli K1 strains at 15 degrees C prevents K1 polysaccharide synthesis (F. A. Troy and M. A. McCloskey, J. Biol. Chem. 254:7377-7387, 1979). Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C. The early expression and resultant morphology of K1 capsular antigen was monitored in temperature upshift experiments by using electron microscopy. Morphological stabilization of the capsule was achieved by treatment of cells with an antiserum specific for the alpha, 2-8-linked polysialosyl antigen. The kinetics of K1 capsule expression in growing cells was measured by bacteriophage adsorption with phage K1F, which required the K1 capsule for binding. The results of temperature upshift experiments showed that capsule first appeared on the cell surface after 10 min. Subsequent bacteriophage binding increased linearly with time until a fully encapsulated state was reached 45 min after upshift. The initiation of K1 capsule appearance was dependent on protein synthesis and the addition of chloramphenicol before temperature upshift prevented any expression of the K1 antigen. Chloramphenicol reduced the rate of K1 synthesis when added after temperature upshift. We conclude from these results that protein synthesis is a prerequisite for activation of capsule expression in vivo, but not for subsequent elongation of polysialosyl chains.

Antibodies↗