K1 Polysaccharide Research Articles

Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria.

Methods were developed for the polyacrylamide gel electrophoretic analysis of capsular polysaccharides of bacteria with Escherichia coli K1 as a model. Conditions were determined for the rapid and gentle extraction of the K1 polysaccharide by incubation of the bacteria in a volatile buffer and for the subsequent removal of the putative phospholipid moiety attached to the reducing end of the polysaccharide. Detection of the polysaccharides after gel electrophoresis was carried out by fluorography of samples labeled by sodium borotritiide reduction or by combined alcian blue and silver staining. The smallest components could be detected only by fluorography, owing to diffusion during staining. Components of the E. coli K1 polysialic acid capsule ranging from monomers to 80 sialic-acid-unit-containing polymers could be separated as distinct bands in a ladderlike pattern. A maximum chain length of 160 to 230 sialyl residues was estimated for the bulk of the K1 polysaccharide from the nearly linear reciprocal relationship between the logarithm of the molecular size and the distance of migration. Gel electrophoresis of capsular polysaccharides of other bacterial species revealed different electrophoretic mobilities for each polysaccharide, with a ladderlike pattern displayed by the fastest-moving components. There are many potential applications of this facile method for the determination of the sizes of molecules present in a polydisperse polysaccharide sample. When combined with the simple method for the isolation of the capsule, as in the case of the K1 capsule, it provides an efficient tool for the characterization and comparison of the capsular polysaccharides of bacteria.

Translocation of capsular polysaccharides in pathogenic strains of Escherichia coli requires a 60-kilodalton periplasmic protein.

An 11.6-kilobase (kb) region of a 34-kb fragment of Escherichia coli DNA that encodes the K1 capsular polysaccharide genes is necessary for translocation of the K1 polysaccharide to the bacterial cell surface. This 11.6-kb region contains a gene, kpsD, encoding a 60-kilodalton protein. The kpsD gene was localized to a 2.4-kb PstI-BamHI fragment. Cells harboring a Tn1000 insertion in kpsD did not synthesize the 60-kilodalton protein and did not express polysaccharide on the cell surface. Immunodiffusion and rocket immunoelectrophoresis of cell extracts, however, demonstrated that K1 polysaccharide was synthesized by these cells. We present evidence that the kpsD gene product is synthesized as a precursor and that the processed form is located in the periplasmic space. Analysis of alkaline phosphatase activity of a kpsD-phoA fusion demonstrated that kpsD expression was under positive regulation. A 260-base-pair AluI fragment located within the kpsD coding sequence was used as a probe and was found to hybridize to chromosomal DNA from E. coli that synthesizes the K2, K5, K7, K12, and K13 capsular polysaccharides but not K3 and K100. These results suggest that the kpsD gene product may be required for export not only of K1 but for other K antigens as well.

The structure of the neuraminic acid-containing capsular polysaccharide of Escherichia coli serotype K9

The acidic capsular polysaccharide isolated from Escherichia coli O9:K9:H12 was investigated by using n.m.r. spectroscopy, methylation analysis, periodate oxidation, and bacteriophage-borne enzyme degradation. The polysaccharide, the structure of which is shown below, is the third E. coli capsular polysaccharide reported to contain neuraminic acid, the others being the K1 and K92 polysaccharides, and it is the first in the E. coli series shown to contain a 4-linked neuraminic acid unit.

Influence of beta-lactam antibiotics and ciprofloxacin on cell envelope of Escherichia coli.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the quantitative composition of the outer membrane (OM) of two strains of Escherichia coli, on lipid translocation into the OM, and on the production of capsular K1 polysaccharide were studied. The phospholipid/amino acid ratio was reduced in almost all OM preparations from antibiotic-treated bacteria. In one strain, antibiotic treatment increased the lipopolysaccharide/amino acid ratio. The amount of peptidoglycan fragments bound to the OM was increased by all the antibiotics. In pulse-chase experiments with a radioactive lipid precursor, ciprofloxacin, imipenem, and aztreonam inhibited phospholipid translocation into the OM. Furthermore, imipenem, cephaloridine, and ciprofloxacin induced a pronounced reduction of the production of capsular K1 polysaccharide. Thus, antibiotics seem to induce marked changes of the quantitative composition of the cell envelope of E. coli. Possible connections of these data with findings on the influence of antibiotics on functional parameters of the host-parasite relationship such as OM immunogenicity and serum resistance are discussed.

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.

EFFICACY OF VARIOUS MONOCLONAL ANTIBODIES (Ab) AGAINST E. COLI (EC)

The morbidity and mortality associated with neonatal EC infections have remained significant despite advances in antimicrobial chemotherapy. To investigate the feasibility of immunotherapeutic approaches, we evaluated 3 different murine monoclonal IgM Ab against a K1 EC strain in the newborn rat model: (1) Ab to group B meningococcal carbohydrate (K1 Ab), (2) Ab to the LPS of the same K1 EC(LPS Ab), and (3) Ab to lipid A. At 5 days of age, each litter was divided into 4 groups. Each group received intraperitoneally one of the above or murine IgM monoclonal Ab to the LPS of P.aeruginosa (control) in a dose of 50 ul/10 gm and subcutaneously EC (LD100). Blood and CSF specimens were obtained daily and mortality recorded for 5 days. Results are summarized as protective efficacy against death. K1 and LPS Ab provided 100% protection neat and at 10-1. All control animals died. These studies indicate that monoclonal Ab to K1 polysaccharide and Ab to the LPS of EC are highly protective against K1 EC and combinations of these two further enhance the efficacy.

Enhanced virulence effect of K1 polysaccharide in neonatal rats challenged withE. coli

Purified K1 polysaccharide enhanced the virulence of non-K1Escherichia coli species when given by orogastric feeding to neonatal rats. Neonatal rats developedE. coli bacteremia when K1 polysaccharide was given concomitantly with non-K1E. coli, whereasE. coli bacteremia did not develop when non-K1E. coli was given alone.E. coli K1 species did cause bacteremia and meningitis when fed to neonatal rats. The mechanism by which k1 polysaccharide enhances virulence can be studied with this model of bacteremia development in neonatal rats.

Influence of growth temperature of Escherichia coli on K1 capsular antigen production and resistance to opsonization.

When Escherichia coli strains that produce K1 capsular polysaccharide antigen at 37 degrees C were grown at 22 degrees C, K1 antigen was not detected in the supernatant or washed-cell fraction of broth cultures. Significant amounts of K1 polysaccharide were detected only when the organism was grown at temperatures of 30 degrees C or higher. Rabbits immunized with an E. coli K1 strain (serotype O18ac:K1:H7) grown at 37 degrees C produced agglutinating antibody to somatic antigen and precipitating and agglutinating antibody to capsular K1 antigen; those immunized with this strain grown at 22 degrees C produced antibody to somatic antigen, but not to K1 antigen. Antibody to somatic antigen was markedly reduced by adsorption with the organism grown at 22 degrees C, while antibody to capsular antigen was not. E. coli K1 strains grown at 37 degrees C (K1 present) resisted phagocytosis and killing if they were opsonized solely by the alternative complement pathway (ACP) using magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid-chelated serum. When these strains were grown at 22 degrees C (K1 absent), they were opsonized efficiently by the ACP (28 versus 94% killing, respectively; P less than 0.001). In addition, a non-K1 mutant of an E. coli K1 strain was opsonized efficiently by the ACP although its encapsulated K1 parent was not. Sensitivity of E. coli strains to the bactericidal activity of serum was observed in strains with and without K1 capsular antigen. These studies demonstrated that production of K1 polysaccharide antigen was regulated by environmental temperature and that K1 capsule plays an essential role in rendering the organism resistant to opsonization by the ACP.

Evaluation of immunotherapeutic approaches for the potential treatment of infections caused by K1-positive Escherichia coli.

Levels of antibody to the K1 polysaccharide capsule were examined in sera from patients naturally infected with K1-positive Escherichia coli, in sera from volunteers vaccinated with a group B meningococcal vaccine, in immune globulin prepared for intravenous use, and in a preparation of murine IgM monoclonal antibody to group B meningococci. In a phagocytic assay, the monoclonal antibody in mouse ascites fluid killed K1-positive E. coli to a final dilution of 1:250,000 (48 ng of antibody protein/ml); no killing was observed with any of the other antibody sources. This monoclonal antibody, which required human complement and exhibited a prozone when high concentrations of antibody (greater than 6 micrograms/ml) were used, killed all K1-positive strains but none of the K1-negative strains of E. coli tested. Levels of K1-binding antibody in the sera of vaccinated volunteers exceeded antibody levels resulting from natural infection or present in commercially prepared immunoglobulin and were less than those obtained in mouse ascites fluid containing the monoclonal antibody.

Endo-N-acetylneuraminidase associated with bacteriophage particles.

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.

Protection against Escherichia coli K1 infection in newborn rats by antibody to K1 capsular polysaccharide antigen.

The protective value of antibody to the K1 capsular polysaccharide antigen of Escherichia coli was investigated in a newborn rat model of E. coli K1 infection. Pregnant rats were immunized intravenously with E. coli, and the agglutinating titer to meningococcal group B polysaccharide, which is identical to K1 polysaccharide, was measured in the serum of rats and their offspring. Convalescent serum from rat mothers showed an increased antibody titer in animals injected twice but not once with E. coli K1. Although no agglutinating antibody was detected in the serum of rat pups, animals suckled by mothers having a meningococcal group B agglutinating titer of 1:8 or greater had reduced infection and mortality rates after intraperitoneal injection with E. coli K1 compared with animals suckled by mothers having a low titer of agglutinating antibody (P less than 0.05). In addition, greater protection could be conferred on rat sucklings by oral supplementation with a horse serum rich in antibody to meningococcal group B polysaccharide, suggesting that antibody was abosorbed from the gastrointestinal tract and by itself could be protective. These studies demonstrated that antibody to the capsular polysaccharide of E. coli K1 altered the severity of E. coli K1 infection. Final clearance of bacteria from the blood appeared to await the maturation of other host defense systems in the newborn rat.

Capsular K1 polysaccharide of Escherichia coli: relationship to virulence in newborn rats and resistance to phagocytosis

The virulence of Escherichia coli strains for newborn rats was related to opsonic requirements of the strains, sensitivity to the bactericidal activity of serum, and K1 capsular polysaccharide content. K1 E. coli strains were more virulent than non-K1 strains after intraperitoneal injection in newborn rats (P less than 0.05) and were more resistant to phagocytosis than non-K1 strains when the classical complement pathway was blocked with Mg-ethyleneglycoltetraacetic acid (P less than 0.0005). Sensitivity to the bactericidal activity of serum was similar among K1 and non-K1 E. coli strains. Two groups of K1 E. coli strains were defined on the basis of opsonic requirements. Group I strains were efficiently opsonized by the alternative complement pathway, while group II strains required the classical complement pathway for opsonization. Group I strains had less detectable K1 polysaccharide in the washed whole cell fraction than group II strains (10.3 versus 18.9 microgram of K1 polysaccharide per 10(10) colony-forming units) and were less virulent than group II strains (mortality, 44 versus 77%, P less than 0.05). The K1 capsular polysaccharide appears to play an important role in determining virulence in newborn rats and opsonic requirements of these strains, but does not contribute to the sensitivity of strains to the bactericidal activity of serum.

Form variation in Escherichia coli K1: determined by O-acetylation of the capsular polysaccharide.

The chemical basis for the alternating antigenic change called form variation noted for the Escherichia coli K1-capsular polysaccharide has been shown by 13C nuclear magnetic resonance to be a result of random O-acetylation of C7 and C9 carbons of the alpha-2-8-linked sialic acid homopolymer. A serologic method (antiserum agar) was developed to identify and isolate the form variants. The O-acetyl positive and O-acetyl negative K1 polysaccharides had unique biochemical and immunologic properties. The O-acetyl-positive variants resisted neuraminidase hydrolysis in contrast to the susceptibility of the O-acetyl negative variant to this enzyme. In addition, O-acetylation altered the antigenicity of the O-acetyl polysaccharides. When injected as whole organisms, O-acetyl positive organisms produced anti-K1 -antibodies in rabbits specific for this polysaccharide variant. O-acetyl negative organisms were comparatively less immunogenic; however, antibodies induced by these organisms reacted with both K1 polysaccharide variants. Burros, injected with either variant, produced antibodies reactive with both K1 polysaccharides.

Interaction of E. coli strains with human serum: Lack of relationship to K1 antigen

Twenty-eight strains of E. coli isolated from infants were compared with respect to opsonic requirements, sensitivity to serum, and ability to activate serum chemotactic factors. Six of the strains were isolated from stools of healthy newborn infants; 22 were isolated from the cerebrospinal fluid or blood of infants with meningitis and/or septicemia. Eighteen of the strains had K1 polysaccharide antigen. Fourteen of the strains (seven with K1 antigen) activated complement via the alternative pathway and all of these strains were well opsonized in 4% pooled human serum. A higher concentration of serum was necessary to opsonize 12 of the 14 strains that did not activate the alternative pathway. A wide variation was also found in opsonic requirements of E. coli strains isolated from healthy and sick infants. There was no relationship of the K1 antigen to opsonic requirements, to capacity to activate complement via the alternative pathway, to generation of chemotactic factors, or to sensitivity to serum cidal activity. Therefore, the association of E. coli with K1 antigen and neonatal meningitis did not appear to be related to these bacteria-serum interactions.