Effects of antibody isotype and host cell type on in vitro neutralization of Chlamydia trachomatis

Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to IgG1, neutralization of C. trachomatis by MAb E-21 in HeLa cells is also corroborative evidence for the role of Fc gamma RIII receptors in this interaction. A fivefold enhancement of infectivity was seen when 10 and 1 micrograms of MAb E-4 per ml were tested in a CI assay with HeLa cells. In performing CI neutralization synergy studies in HeLa cells with MAbs E-4 and E-21, antagonism between MAbs E-4 and E-21 was observed at MAb E-4 concentrations of 10 and 1 micrograms/ml for all concentrations of MAb E-21 tested (10 to 0.1 micrograms/ml). When HaK cells were used in the same studies, no antagonism between the MAbs was found. In addition, when HeLa cells were used in a CI assay, polyclonal serum raised to a peptide representing variable domain IV of the major outer membrane protein inhibited the neutralizing ability of MAb E-21. The blocking of neutralization and the enhancement of infectivity by chlamydia-specific antibodies seen in this investigation with HeLa cells may have important clinical implications for developing preventive strategies for chlamydial infections.     

Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.     

Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae

The periplasmic invertase was purified from Saccharomyces cerevisiae och1::LEU2 disruptant cells (delta och1), which have a defect in elongation of the outer chain attached to the N-linked core oligosaccharides (Nakayama, K., Nagasu, T., Shimma, Y., Kuromatsu, J., and Jigami, Y. (1992) EMBO J. 11, 2511-2519). Structural analysis of the pyridylaminated (PA) neutral oligosaccharides released by hydrazinolysis and N-acetylation confirmed that the och1 mutation causes a complete loss of the alpha-1,6-polymannose outer chain, although the PA oligosaccharides (Man9GlcNAc2-PA and Man10GlcNAc2-PA), in which one or two alpha-1,3-linked mannose(s) attached to the endoplasmic reticulumn (ER)-form core oligosaccharide (Man8GlcNAc2) were also detected. Analysis of the delta och1 mnn1 strain oligosaccharides released from total cell mannoprotein revealed that the delta och1 mnn1 mutant eliminates the alpha-1,3-mannose attached to the core and accumulates predominantly a single ER-form oligosaccharide species (Man8GlcNAc2), suggesting a potential use of this strain as a host cell to produce glycoproteins containing mammalian high mannose type oligosaccharides. The delta och1 mnn1 alg3 mutants accumulated Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete corelike oligosaccharide and the leaky phenotype of the alg3 mutation. All the results suggest that the OCH1 gene encodes an alpha-1,6-mannosyltransferase that is functional in the initiation of alpha-1,6-polymannose outer chain addition to the N-linked core oligosaccharide (Man5GlcNAc2 and Man8GlcNAc2) in yeast.     

Surgical treatment for the recurrence of colorectal cancer

Analysis by two-dimensional gel electrophoresis of the N-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes of L-[35]S-cysteine-labeled elementary bodies of Chlamydia pneumoniae strain IOL-207, Chlamydia trachomatis serovar LGV2, D, and F, and Chlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein of C. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of the C. trachomatis and C. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteine-rich protein of C. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the low-molecular-mass (12-15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in each individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the low-molecular-mass cysteine-rich protein.     

Detection of Chlamydia trachomatis antibodies by 2 novel tests: rELISA and peptide EIA

The performance of 2 newly developed enzyme immunoassays (EIAs) intended for the routine serological diagnosis of chlamydial infections was evaluated. rELISA is based on a recombinant lipopolysaccharide antigen which detects chlamydia genus-specific antibodies, and Chlamydia trachomatis EIA is based on a peptide derived from major outer membrane protein and is therefore species-specific. Both tests distinguished patients with tubal factor infertility (TFI) or pelvic inflammatory disease (PID) from the controls. The prevalence of IgA antibodies was higher for the PID patients than for the TFI patients; the finding indicates a more active state of infections for the PID patients. Furthermore, C. trachomatis EIA detected more IgG antibodies in the TFI patients than in patients with non-tubal factor infertility. In conclusion, rELISA detected chlamydial antibodies in general, and C. trachomatis EIA detected species-specific antibodies. These EIA tests may be useful in the serodiagnosis of chlamydial infection.     

Workshop on in vitro neutralization of Chlamydia trachomatis: summary of proceedings

A task force evaluated an in vitro antibody-mediated chlamydial neutralization assay for its utility as a method to assess functional correlates of antibody responses to Chlamydia trachomatis. Two monoclonal antibodies that recognize different major outer membrane protein (MOMP) epitopes for a C. trachomatis serovar B strain exhibit good in vitro neutralizing activity, with a maximum of 90% neutralization. Calculations based on the 50% neutralization point indicated that 100% neutralization could theoretically be achieved when only 10% of the MOMP molecules bound antibody. Monoclonal antibodies that recognized either a heterologous MOMP or the genus-specific chlamydial lipopolysaccharide did not produce neutralizing activity. The standardized assay will be useful to establish if in vitro neutralizing antibody responses are predictive of protective immunity and will aid in defining chlamydial antigens and epitopes that may be attractive vaccine candidates.     

Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation

The synthesis of oligosaccharide-lipids thought to play a role in the attachment of carbohydrate to protein has been studied in incubations of slices from calf kidney, pancreas, thymus, and liver, as well as from hen oviduct. These compounds were characterized after radiolabeling of their saccharide moiety by incubation with [14C]glucose or [14C]mannose and a comparison was made with the oligosaccharide-lipid produced by thyroid slices. Furthermore, the unlabeled glycolipid was prepared from hen oviduct for the purpose of quantitating its sugar constituents. Purification of the oligosaccharide-lipids extracted with chloroform/methanol/water (10/10/3) was achieved by DEAE-cellulose chromatography and their carbohydrate moieties were released by mild acid hydrolysis. On the basis of gel filtration it was determined that the lipid-bound oligosaccharides formed by oviduct, thymus, kidney, and liver had molecular weights comparable to that from thyroid (about 2400). The saccharide moiety of the glycolipid from pancreas was however distinctly smaller in size with a molecular weight of approximately 1800. Analyses of the radiolabeled oligosaccharide-lipids from oviduct, kidney, and thymus indicated that they, like the compound from thyroid slices, but unlike those believed to be formed by cell-free systems from various tissues, contained glucose in addition to mannose and N-acetylglucosamine as their monosaccharide constituents. This compositional data was supported by the finding that the unlabeled oligosaccharide from oviduct consists of 10 mannose, 1 glucose, and 2 N-acetylglucosamine residues. Sodium borohydride reduction of this oviduct saccharide moiety indicated that 1 of the 2 glucosamines was situated in a reducing terminal position. The radiolabeled oligosaccharide from the glycolipid produced by pancreas differed from the others analyzed in that it contained only trace amounts of glucose. Upon treatment with alpha-mannosidase this glucose-deficient pancreatic oligosaccharide was extensively digested (85% of the mannose released). In contrast, the carbohydrate moieties of oviduct, kidney, and thymus, like that of thyroid, underwent a more limited digestion with the alpha-mannosidase (55% or less of the mannose released) suggesting that the presence of glucose may serve to block a more complete degradation of these oligosaccharides by this enzyme.     

Sulfated polysaccharides and a synthetic sulfated polymer are potent inhibitors of Chlamydia trachomatis infectivity in vitro but lack protective efficacy in an in vivo murine model of chlamydial genital tract infection

Heparin, dextran sulfate, pentosan polysulfate, and a sulfated synthetic copolymer of acrylic acid and vinyl alcohol were shown to be potent inhibitors of Chlamydia trachomatis infectivity for cultured human epithelial cells. Despite their potent antichlamydial activity in vitro, neither heparin nor dextran sulfate was effective in inhibiting the infectivity of C. trachomatis in a murine model of chlamydial infection of the female genital tract.     

Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR

A nested PCR for genus-specific amplification of the Chlamydia omp1 locus was established. This PCR detected single template molecules in 200-microl specimen aliquots. Amplified chlamydial omp1 alleles were typed by heminested species PCRs and allele PCRs. We applied this method to 407 specimens from several host animals with various clinical conditions, and we detected prevalences of chlamydiae from 6 to 50%. Amplicons from peacock enteritis and equine infertility specimens were not typeable according to present omp1 allelic criteria for the chlamydial species. DNA sequencing revealed novel omp1 alleles which were 29.9 and 47.6% divergent in the deduced peptide sequences from the most closely related chlamydiae. Phylogenetic reconstruction indicated segregation of these alleles from the current four chlamydial species (90 and 97% bootstrap support), thus strongly suggesting the existence of additional chlamydial species. Allele typing of amplicons from swine with intestinal, urogenital, and respiratory infections demonstrated several unique omp1 allelic variants of Chlamydia trachomatis. These novel alleles had deduced peptide sequences which were 11.6 to 19% divergent from porcine C. trachomatis S45. Mutations were clustered in the C-terminal region of variable segment IV of the omp1 locus encoding subspecies and serovar determinants of the chlamydial major outer membrane protein, thus implying that there are numerous serovars of porcine C. trachomatis. These results demonstrate the need for routine application of sensitive genus-specific detection of chlamydiae in animal specimens and suggest a more prominent role than anticipated for chlamydiae in animal diseases.     

High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

Lack of correlation between the detection of Chlamydia trachomatis DNA in synovial fluid from patients with a range of rheumatic diseases and the presence of an antichlamydial immune response

OBJECTIVE: To resolve how frequently Chlamydia trachomatis and Chlamydia pneumoniae DNA are present in the joints of unselected patients with reactive arthritis (ReA) and undifferentiated oligoarthritis, and to determine if there is an accompanying serologic or cellular antichlamydial immune response. METHODS: Two polymerase chain reaction (PCR) protocols to detect the plasmid of C. trachomatis and the outer membrane protein 1 gene of C. pneumoniae were developed for specific use with synovial fluid (SF). Subsequently, the assays were used to detect DNA from the 2 organisms in SF from 54 adult patients with rheumatic diseases, including 4 with sexually acquired ReA and 31 with undifferentiated oligoarthritis. The presence of chlamydial antibodies and SF lymphocyte proliferation responses were determined in parallel. RESULTS: The PCR protocols were species-specific and highly sensitive. SF samples from 15 patients (8 with undifferentiated oligoarthritis, 3 with ReA, 1 with rheumatoid arthritis, and 1 with psoriatic arthritis) were positive for C. trachomatis. There was no significant correlation between the presence of C. trachomatis DNA in the joint and a Chlamydia-specific synovial T cell response or a serologic response. C. pneumoniae was not detected in any of the 54 patients, although it was identified in the SF from a rheumatoid arthritis patient outside this study, demonstrating that the assay was capable of detecting the organism in the joint. CONCLUSION: C. trachomatis DNA was present in ReA patients and in nearly one-third of unselected patients with undifferentiated oligoarthritis, which further supports the hypothesis that it plays an important role in disease pathogenesis. However, its presence did not correlate with evidence of an antichlamydial immune response. Despite previous anecdotal reports, C. pneumoniae does not appear to be a major cause of undifferentiated oligoarthritis or ReA.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia-containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia-containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.     

Proteolytic release and partial characterization of human sperm-surface glycopeptides

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of O- and N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface.     

Post-myocardial infarct rupture of the interventricular septum. The prognostic factors of hospital mortality

Rod-like projections on the surface of Chlamydia trachomatis have been studied by a combination of computer image analysis and electron microscopy. The rods, c. 60-80 A in diameter and c. 500 A in length, were found on the surface of prokaryocells of C. trachomatis inserted in the cytoplasmic membrane through a ring-like structure in the outer membrane. The rod-like structures were found at all stages of the life cycle, even in very small elementary bodies (EBs) of C. trachomatis and in vesicles < 0.2 micron. Computer image analysis of isolated rods indicated that they comprise helically arranged subunits with a periodicity of c. 50 A. From their localisation and distribution, these structures may be related to the proliferation, or to the infectivity, of chlamydiae.     

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.     

Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins

Iron is a well-established mediator of virulence in several bacterial pathogens, yet little is known about the role of iron in infectious disease processes caused by obligate intracellular bacterial pathogens. In this study, the effect of iron limitation was examined for the sexually transmitted infectious agent Chlamydia trachomatis in an in vitro model of human genital infection using the intracellular iron-chelating reagent deferoxamine mesylate (Desferal). Iron restriction caused a significant reduction in infectivity of C. trachomatis elementary bodies (EB) harvested from Desferal-exposed polarized epithelial cells when compared to that of EB harvested from iron-sufficient control cell cultures. Replacement of the Desferal exposure medium with medium containing iron-saturated transferrin restored chlamydial infectivity, whereas replacement with growth medium alone had no effect. The following three prominent morphological features were observed by electron microscopic examination of chlamydia-infected cells exposed to Desferal: (i) inclusions containing chlamydiae greatly delayed in maturation, (ii) substantial blebbing within chlamydial inclusions, and (iii) electron-dense material surrounding inclusions. Protein analyses of highly purified EB by two-dimensional polyacrylamide gel electrophoresis revealed that there were at least 19 candidate iron-repressible proteins in C. trachomatis and at least one protein which was iron inducible. One putative iron-repressible protein was confirmed by Western blot (immunoblot) analysis to be the chlamydial heat shock protein 60 (hsp60). The enhanced production of this antigen by chlamydiae as a result of iron limitation is of particular importance since there is a well-documented association between chlamydial hsp60 and destructive immunopathological sequelae in infected patients.     

Structural changes in the oligosaccharide moiety of human IgG with aging

In order to elucidate the relationship between glycosylation of IgG and aging, oligosaccharide structures of human IgG purified from sera of men and women aged 18 to 73 years were investigated. Oligosaccharides were liberated quantitatively from IgG by hydrazinolysis followed by N-acetylation and were tagged with p-aminobenzoic acid ethyl ester. The oligosaccharide structures were then analyzed by HPLC in conjunction with sequential exoglycosidase digestion. All IgG samples were shown to contain a series of biantennary complex type oligosaccharides which consisted of +/-Galbeta1-4GlcNAcbeta1-2Manalpha1-6(+/-GlcNAcbeta 1-4)(+/-Galbeta1-4GlcNAcbeta1-2Man(alpha)1-3)Man(beta)1-+ ++4GlcNAcbeta1-4(+/- Fucalpha1-6)GlcNAc and their mono- and disialo glycoforms in different ratios. In female IgG samples only, the incidence of non-galactosylated oligosaccharides with non-reducing terminal GlcNAc residues increased with aging (r>0.8), whereas that of digalactosylated oligosaccharides decreased (r<-0.8). A weaker correlation was observed between aging and the incidence of neutral and monosialo oligosaccharides in female IgG (r=0.461 and r= -0.538, respectively) and between aging and the incidence of oligosaccharides with a bisecting GlcNAc in both male and female IgG samples (r=0.566 and r=0.440, respectively). In addition, a significant change with aging in the galactosylation of IgG oligosaccharides was observed in females in their thirties, fifties, and sixties (p<0.02, p<0.01, and p<0.04, respectively). These findings may contribute to our understanding of autoimmune diseases such as rheumatoid arthritis in which glycosylation is involved.     

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.     

Identification of T cells that respond to serovar-specific regions of the Chlamydia trachomatis major outer membrane protein in persons with serovar E infection

Protection from Chlamydia trachomatis infection is serovar-specific and T cell-dependent; however, the T cell epitopes identified to date have not been serovar-specific. The chlamydial major outer membrane protein (MOMP) contains serovar-specific B cell epitopes in four regions of the molecule whose amino acid sequence varies among serovars, the variable segments (VS). Serovar-specific T cell epitopes were sought by examining proliferation of blood mononuclear cells (PBMC) from Chlamydia-infected patients in response to VS peptides of serovar E MOMP. Serovar E-specific peptides from VS1, VS2, and VS4 stimulated PBMC to a greater extent in serovar E-infected than in non-E-infected subjects. Peptides containing constant regions of MOMP were recognized equally by all infected persons. The observed responses were attributable to T cells. T cell recognition of serovar-specific regions of MOMP is common and may contribute to the serovar-specific protection previously observed.