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Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis , the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis . Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/β-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through β-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/β-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear β-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

Recently, macrophages were shown to be capable of differentiating toward two phenotypes after antigen stimulation: a classically activated (M1) or an alternatively activated phenotype (M2). To investigate the effect of Salmonella enteric serovar typhimurium (S. typhimurium) on macrophage differentiation, we compared macrophage phenotypes after infection of murine bone marrow-derived macrophages with wild-type S. typhimurium and its isogenic rfc mutant. S. typhimurium C5 induced M1 macrophage polarization and enhanced inducible nitric oxide synthase expression by macrophages; this induction was dependent on Toll-like receptor 4. In contrast, the Δrfc mutant (S. typhimurium C5 rfc::Km(r)) lost this function and induced an M2 response in the macrophages. Here, we propose that S. typhimurium C5 is capable of polarizing macrophages towards the M1 phenotype and that this polarization is dependent on the O antigen encoded by rfc. Our finding indicates that M1 macrophage polarization induced by S. typhimurium may be related to the ability of this intracellular bacterium to survive and replicate within macrophages, which is essential for systemic disease.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate beta-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer's patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10(6) higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1x10(10)CFU) or i.p. (1x10(7) CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD(50) (5x10(6) CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.

Purpose: Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples., Experimental Design: Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage-inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions., Results: C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers., Conclusions: We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.

Streptococcus mutans utilizes sucrose to synthesize glucans by glucosyltransferase and fructans by fructosyltransferase (FTF). Antibodies raised against a recombinant FTF were used to study S. mutans FTF secretion. Low amounts of cell-free FTF were found in culture of S. mutans grown with sucrose, while an increase in bacteria displaying cell surface FTF was detected. FTF added to S. mutans cultures was adsorbed to bacteria grown with sucrose but not to bacteria grown with glucose or fructose or to a gtf inactivated mutant grown with sucrose. Recombinant FTF was found to have high affinity for glucans suggesting that fructans and glucans are an integral part of the polysaccharide matrix of oral biofilms.

Core 2 O-glycans terminated with sialyl-Lewis x (sLe(X)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLe(X) extended from a core 2 branch (C2-O-sLe(X)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLe(X) was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(X) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(X)-related glycans. Unlike anti-sLe(X) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both.

FtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.

Two synthetic peptides 31 and 32 amino acids in length were prepared as deduced from a known amino acid sequence of penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus. Two monoclonal antibodies were generated from fused cells of myeloma cells and splenic cells of mice immunized with the synthetic peptides. Western blot (immunoblot) analysis demonstrated specific binding of the antibodies to PBP2' of a methicillin-resistant S. aureus strain. An immunoradiometric assay was developed by using these antibodies for simple detection of PBP2'.

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2' of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin-susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2' was developed using these antibodies. The method could detect PBP2' extracted from as few as 3 x 10(4) cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio-count was observed. IRMA was positive for all 51 methicillin-resistant staphylococci isolated from patients, and was negative for all the 28 methicillin-susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.

The structural gene for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus was cloned and sequenced. The nucleotide sequence of the 2,458-bp chromosomal fragment was determined, and the 2,148-bp coding region for PBP2 was identified. Determination of ten N-terminal amino acids of the PBP2 protein indicated that N-terminal methionine had been removed from the primary translational product. Thus, PBP2 is comprised of 715 amino acids with a molecular mass of 79,147. Nucleotide sequences having some homology with the PBP2 gene and proteins cross-reactive with anti-PBP2 antibody were detected in some other species of staphylococci by polymerase chain reaction and Western blot analysis, respectively.

The pbpB gene, which encodes penicillin-binding protein (PBP) 2B of Bacillus subtilis, has been cloned, sequenced, mapped, and mutagenized. The sequence of PBP 2B places it among the class B high-molecular-weight PBPs. It appears to contain three functional domains: an N-terminal domain homologous to the corresponding domain of other class B PBPs, a penicillin-binding domain, and a lengthy carboxy extension. The PBP has a noncleaved signal sequence at its N terminus that presumably serves as its anchor in the cell membrane. Previous studies led to the hypothesis that PBP 2B is required for both vegetative cell division and sporulation septation. Its sequence, map site, and mutant phenotype support this hypothesis. PBP 2B is homologous to PBP 3, the cell division protein encoded by pbpB of Escherichia coli. Moreover, both pbpB genes are located in the same relative position within a cluster of cell division and cell wall genes on their respective chromosomes. However, immediately adjacent to the B. subtilis pbpB gene is spoVD, which appears to be a sporulation-specific homolog of pbpB. Inactivation of SpoVD blocked synthesis of the cortical peptidoglycan in the spore, whereas carboxy truncation of PBP 2B caused cells to grow as filaments. Thus, it appears that a gene duplication has occurred in B. subtilis and that one PBP has evolved to serve a common role in septation during both vegetative growth and sporulation, whereas the other PBP serves a specialized role in sporulation.

A high-molecular-weight band has been detected in Western immunoblots of nonboiled Escherichia coli samples incubated with polyclonal antiserum against penicillin-binding protein 1B (PBP 1B). This band was shown to be a dimer of PBP 1B. The dimer was more strongly associated with the envelope than the monomer, and it was still able to bind penicillin G. Analysis of the binding of fusion proteins of PBP 1B and beta-lactamase showed that the part of PBP 1B necessary for complex formation lies in the amino-terminal half of the protein.

Enterococcus hirae S185, a clinical isolate from swine intestine, exhibits a relatively high resistance to penicillin and contains two 77-kDa penicillin-binding proteins 3 of high (PBP 3s) and low (PBP 3r) affinity to penicillin, respectively. A laboratory mutant S185r has been obtained which overproduces PBP 3r and has a highly increased resistance to penicillin. Peptide fragments specifically produced by trypsin and SV8 protease digestions of PBP 3r were isolated, and the amino acid sequences of their amino terminal regions were determined. On the basis of these sequences, oligonucleotides were synthesized and used as primers to generate, by polymerization chain reaction, a 233-bp DNA fragment the sequence of which translated into a 73-amino-acid peptide segment of PBP 3r. These structural data led to the conclusion that the E. hirae PBP 3r and the methicillin-resistant staphylococcal PBP 2' are members of the same class of high-Mr PBPs. As shown by immunological tests, PBP 3r is not related to PBP 3s but, in contrast, is related to the 71-kDa PBP 5 of low penicillin affinity which is responsible for penicillin resistance in E. hirae ATCC 9790 and R40.

Monoclonal antibodies (MAbs) against four different antigenic determinants of penicillin-binding protein (PBP) 1b were used to study the transglycosylase and transpeptidase activities of PBP 1b. Enzyme kinetics in the presence of and without the MAbs were determined, and the synthesized murein was analyzed. Two MAbs against the transglycosylase domain of PBP 1b appeared to inhibit this reaction. One MAb inhibited only the transpeptidase reaction, and one inhibited both enzymatic activities of PBP 1b. The latter two MAbs bound to the transpeptidase domain of PBP 1b. The following major conclusions were deduced from the results. (i) Transpeptidation is the rate-limiting step of the reaction cascade, and it is dependent on the product of transglycosylation. (ii) PBP 1b has only one type of transpeptidase activity, i.e., a penta-tetra transpeptidase activity. (iii) PBP 1b is probably a globular protein which has two intimately associated enzymatic domains.

A method has been developed to study the orientation of proteins in the cytoplasmic membrane of Escherichia coli. Vesicles from sonicated cells were incubated in droplets on electron microscope support grids in sequence with a monoclonal antibody (MAb) against a protein with an unknown orientation (PBP 1b) followed by a MAb against a periplasmic component (peptidoglycan). The different MAbs were made visible with 5- and 10-nm gold-conjugated secondary antibodies, respectively. PBP 1b appeared to colabel with peptidoglycan. The labeling of PBP 1b in membrane vesicles with MAbs against four different epitopes was further used to estimate the number of PBP 1b molecules per cell. Approximately 1,400 PBP 1b molecules per cell grown in broth were labeled. The spatial distribution of the epitopes of the MAbs was studied by immunocolabeling of pairs of MAbs and by competitive antibody-binding inhibition. It could be tentatively concluded that the four epitopes form a cluster of antigenic determinants which occupy less than half of the surface of PBP 1b.

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.

Penicillin-binding proteins (PBPs) from Haemophilus influenzae RD purified by a combination of affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electroelution were used to immunize rabbits to obtain specific antisera. Antisera directed against PBP 1 (1b) of H. influenzae cross-reacted with representative organisms of the family Pasteurellaceae and with many members of the family Enterobacteriaceae but not with other gram-negative organisms. Immunization with purified PBP 3 of H. influenzae produced antisera that reacted with PBP 1 (1b) of H. influenzae and showed the same cross-reactive pattern with other species as the anti-PBP 1 antiserum. A 24,000-molecular-weight polypeptide of H. influenzae, not radiolabeled by [35S]penicillin, reacted with antisera against purified PBPs 1 (1a, 1b), 2, and 3. The results suggest that antigenic epitopes are shared among similar PBPs from related species and even among different PBPs within the same species.

The structural gene for a glucan-binding protein (GBP) of Streptococcus mutans has been inserted into a bacteriophage lambda vector and expressed in Escherichia coli K12. Lysates of E. coli infected with the recombinant phage contain an antigenic protein of the same size as S. mutans GBP. The GBP synthesized in E. coli can be affinity-purified on immobilized glucan and antiserum raised against it has been shown to precipitate fructosyltransferase activity from S. mutans.

The present studies assayed antibody activities in serum and saliva of animals immunized by different routes, with cells of S. mutans or cell-free preparations containing GTF, FTF, LTA and/or dextranase synthesized by S. mutans. The results show that the type of immunogenic preparation and the route of its administration can elicit different antibody response and may in part explain the disparity of results achieved by different investigators. The results further emphasize the need to use standardized preparations and carefully described protocols for vaccination.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

A beta-galactoside alpha 1 leads to 2 fucosyltransferase (H-enzyme) from human group O plasma which provides H blood group specificity to erythrocyte membranes has been purified approximately 22,000-fold by chromatography on DEAE-Sepharose CL-6B, GDP-hexanolamine-Sepharose 4B and SP-Sephadex C-50. The molecular weight of the H-enzyme was estimated to be 150,000 by gel filtration. Human group O erythrocyte membranes which had lost their H blood group activity by the action of alpha 1 leads to 2 fucosidase were fucosylated by the transferase, and restored the H activity. Radioactive L-fucose appeared to be incorporated into glycolipid blood group substances of erythrocyte membranes. The activity of the alpha 1 leads to 2 fucosyltransferase from human plasma, stomach mucosa, erythrocyte membranes and porcine stomach mucosa were specifically inhibited by the rabbit antiserum immunized with the preparation of human plasma H-enzyme. The anti-plasma H-enzyme antiserum did not inhibit the activities of alpha 1 leads to 3 N-acetylgalactosaminyltransferase (A-enzyme), alpha 1 leads to 3 galactosyltransferase (B-enzyme), and beta-N-acetylglucosaminide alpha 1 leads to 3 and alpha 1 leads to 4 fucosyltransferases from human plasma and stomach mucosa.

A demonstration that antiserums from monkeys immunized with fructosyltransferase and glucosyltransferase reduced the activities of the enzymes in producing glucans and levans was done. The nature of the inhibiting factor has not yet been identified. The local immunization of monkeys with glycosidic hydrolases produced a sufficient amount of inhibiting principle, presumably antibody, to inhibit neuraminidase one month after the first immunization and this degree of immunization, which reached degrees of 50%, remained constant throughout 11 months of immunization of these monkeys. The immunizing principle has not yet been found in saliva. A criterion for the feasibility for immunization has been met. A significant reduction in streptoccal glucosyltransferase, fructosyltransferase, and neuraminidase activity was noted after immunization with enzymes.

The genes for fructosyltransferase (ftf) and glucan-binding protein (gbp) of Streptococcus mutans strain Ingbritt have been cloned in E. coli using bacteriophage and plasmid vectors. A single ftf gene appears to be responsible for the appearance of several FTF of different electrophoretic mobilities. Despite the fact that both ftf and gbp proteins synthesise fructans from sucrose in S. mutans and shown closely similar electrophoretic mobilities and isoelectric points, the restriction maps of the two genes are distinct, their DNAs do not hybridise and there is no immunological cross-reaction between the two proteins.

The occurrence of a potent antibody against plasmatic A and B glycosyltransferase activities has been characterized in a patient (blood group A1) transplanted with a bone marrow from a blood group O donor. A and B glycosyltransferases were purified to near homogeneity from plasma of A1 and B blood-group individuals. The half-maximal inhibition of both enzymes was obtained at 1 to 2 micrograms/mL of the post-transplant IgG fraction, prepared by protein A-sepharose chromatography. A and B glycosyltransferases were also recognized by the post-transplant IgG fraction after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrophoretic transfer to nitrocellulose membranes.

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.