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Typhax is an investigational typhoid fever vaccine candidate that is comprised of Vi polysaccharide from Salmonella enterica serovar typhi (S. Typhi) non-covalently entrapped in a glutaraldehyde catalyzed, cross-linked α-poly-L-lysine and CRM 197 protein matrix. A previous Phase 1 trial of an aluminum phosphate adjuvanted Typhax formulation showed it induced Vi IgG after a single dose but that subsequent doses failed to further boost Vi IgG levels. The current study asked whether Advax-CpG adjuvant might instead be able to overcome polysaccharide-induced immune inhibition and improve Typhax immunogenicity. Advax-CpG adjuvanted Typhax elicited high and sustained Vi IgG responses in mice, rabbits and non-human primates (NHP) with levels being boosted by repeated immunization. High Vi antibody responses were lost in CD4 + T cell depleted mice confirming that despite the lack of conjugation of the polysaccharide to the carrier protein, Typhax nevertheless acts in a T cell dependent manner, explaining its ability to induce long-term B cell memory responses to Vi capable of being boosted. In NHP, Advax-CpG adjuvanted Typhax induced up to 100-fold higher Vi IgG levels than the commercial Typhim Vi polysaccharide vaccine. Typhax induced high and sustained serum bactericidal activity against S. Typhi and stimulated robust Vi IgG responses even in animals previously primed with a pure polysaccharide vaccine. Hence Advax-CpG adjuvanted Typhax vaccine is a highly promising candidate to provide robust and durable protection against typhoid fever., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: YHO and NP are affiliated with Vaxine Pty Ltd which hold the rights to Advax adjuvants KK, RTC, AT are affiliated with Matrivax which holds rights to Typhax, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pneumolysin is a highly conserved, cholesterol-dependent cytolysin that is an important Streptococcus pneumoniae virulence factor and an attractive target for vaccine development. To attenuate pneumolysin toxicity, a genetic toxoid was constructed with two amino acid changes, G293S and L460D, termed PLY-D, that reduced cytolytic activity > 125,000-fold. In mice, PLY-D elicited high anti-PLY IgG antibody titers that neutralized the cytolytic activity of the wild-type toxin in vitro. To evaluate the protective efficacy of PLY-D, mice were immunized intramuscularly and then challenged intranasally with a lethal dose of 28 clinical isolates of S. pneumoniae originating from different geographical locations, disease states (i.e. bacteremia, pneumonia), or body sites (i.e. sputum, blood). PLY-D immunization conferred significant protection from challenge with 17 of 20 serotypes (85%) and 22 of 28 strains (79%). Further, we demonstrated that immunization with PLY-D provided statistically significant improvement in survival against challenge with serotype 4 and 18C strains compared to mice immunized with a pneumococcal conjugate vaccine Prevnar 13® (PCV13). Co-administration of PLY-D and PCV13 conferred greater protection against challenge with a serotype 6B strain than immunization with either vaccine alone. These data indicate that PLY-D is a broadly protective antigen with the potential to serve as a serotype-independent vaccine against invasive pneumococcal disease either alone or in combination with PCVs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Matrivax Corporation. Published by Elsevier Ltd.. All rights reserved.)

Background: Typhoid fever remains a significant cause of morbidity and mortality in developing countries especially in children ≤5 years old. Although the widely available unconjugated Vi polysaccharide vaccines are efficacious, they confer limited, short-term protection and are not approved for young children or infants. Vi conjugate vaccines, however, are now licensed in several typhoid endemic countries for use in children >6 months of age. As an alternative to conjugate vaccines, Matrivax has applied its novel 'virtual conjugation' Protein Capsular Matrix Vaccine (PCMV) technology to manufacture Typhax, which is composed of Vi polysaccharide entrapped in a cross-linked CRM197 matrix., Methodology: A randomized, double-blinded, dose escalating Phase 1 study was performed to compare the safety and immunogenicity of three dose levels of aluminum phosphate adjuvanted Typhax (0.5, 2.5, or 10 μg of Vi antigen) to the FDA licensed vaccine, Typhim Vi, and placebo. Groups of 15 healthy adult subjects aged 18 to 55 years were randomized and received Typhax, Typhim Vi, or placebo at a ratio of 9:3:3. Typhax and placebo were administered in a two-dose regimen (Days 0 and 28) while Typhim Vi was administered as a single-dose on Day 0 with a placebo administered on Day 28. All doses were administered as a 0.5 mL intramuscular (IM) injection in a blinded fashion. The anti-Vi IgG antibody response was determined preimmunization (Day 0) and on Days 14, 28, 42, and 180 by ELISA. Seroconversion was defined as a titer 4-fold or greater above baseline., Principal Findings: All Typhax vaccine regimens were well tolerated and adverse events were low in number and primarily characterized as mild in intensity and similar in incidence across the treatment groups. Reactogenicity, primarily pain and tenderness at the injection site, was observed in both the Typhax and Typhim Vi treatment groups; a modest increase in incidence was observed with increasing Typhax doses. Following one dose of Typhax, seroconversion rates at day 28 were 12.5%, 77.8%, 66.7% at the 0.5, 2.5, and 10 μg dose levels, respectively, compared to 55.6% and 0% in the Typhim Vi and placebo groups, respectively. A second dose of Typhax on Day 28 did not elicit a significant increase in GMT or seroconversion at Day 42 or Day 180 at any dose level., Conclusions: Collectively, the results from this randomized phase 1 clinical trial indicate that Typhax is safe, well tolerated, and immunogenic. After a single dose, Typhax at the 2.5 and 10 μg dose levels elicited comparable anti-Vi IgG titers and seroconversion rates as a single dose of Typhim Vi (25 μg dose). A second dose of Typhax at Day 28 did not elicit a booster response., Trial Registration: ClinicalTrials.gov NCT03926455., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Robert Cartee, Ann Thanawastien, Tom Griffin and Kevin Killeen are all employees of Matrivax whereas John Mekalanos is a founder of Matrivax. Stephan Bart was an employee at Accelovance at the time of the Phase 1 trial and is currently affiliated with Trial Professionals Consulting Group Inc. Morningside Ventures was the Venture Capital Group that funded the Phase 1 clinical study.

Typhax is an investigational typhoid fever vaccine candidate that was GMP manufactured applying Protein Capsular Matrix Vaccine (PCMV) technology. It consists of Vi polysaccharide antigen, derived from S . Typhi, non-covalently entrapped in a glutaraldehyde catalyzed cross-linked α-poly-L-lysine and CRM197 protein matrix. Analysis of Typhax determined the average molecular weight of the vaccine particles was approximately 6 x 10 6 Daltons, corresponding to particles containing 1-2 molecules of Vi polysaccharide and 10-20 molecules of CRM197 protein. The ratio of the concentration of Vi to CRM197 protein in Typhax is 2.4:1. Preclinical immunogenicity studies in mice demonstrated that Typhax was immunogenic and elicited a significant increase in anti-Vi IgG antibody titers following each immunization. The anti-Vi IgG antibody response elicited by Typhax in rabbits increased as the dose increased from 0.1 µg to 2.5 µg. Further, at the 2.5 and 10 µg dose levels, the anti-Vi IgG antibody titers increased after the second and third immunizations. At the 10 µg dose level, 100% of rabbits seroconverted. In the non-human primate (NHP) study, 100% seroconversion was observed at both 2.5 µg and 10 µg dose levels after the first immunization. A murine in vivo immunopotency study demonstrated that Typhax stored at 4°C was stable for at least 30 months. Collectively, the Typhax in vitro profile, preclinical immunogenicity studies, and rabbit toxicology study indicate that Typhax is a viable typhoid fever vaccine candidate for Phase 1 clinical trial evaluation.

Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell-independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a "carrier protein" antigen. Such "conjugate vaccines" efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.

The chain length of Streptococcus pneumoniae type 3 capsular polysaccharide (cellubiuronic acid) is tightly regulated by the cellubiuronic acid synthase through an assembly process involving a catalytic motif that is potentially conserved over a wide range of related processive beta-glucan synthases. Cellubiuronic acid is initiated on a lipid and is composed of alternating beta-1,3-Glc and beta-1,4-glucuronic acid (GlcUA) linkages. The entire assembly process is carried out by a polypeptide synthase thought to contain a single active site, suggesting that the donor specificity is controlled by the terminal nonreducing sugar in the acceptor subsite. Shortly after initiation, the synthase undergoes an allosteric transition accompanied by the tight binding of the nascent chain via its nonreducing oligosaccharide terminal segment to the carbohydrate acceptor recognition site. The chain length of polysaccharide assembled by recombinant synthase in Escherichia coli membranes was determined by an ejection mechanism that appeared to be a reversal of the allosteric transition of the synthase from the transitory to the fully processive state. The rates of both ejection and transition were shown to be highly sensitive to the concentration of UDP-GlcUA. As the concentration of UDP-GlcUA was increased, both the rate of synthesis and the processive turnover time increased. The product of the processive turnover time and the rate of synthesis predicted a marked increase in polysaccharide chain size (from 50 to 1150 kDa) over a relatively narrow concentration range of 1-11.5 microm UDP-GlcUA. The kinetic model chain length predictions were in close agreement with chemically determined sizes of polysaccharides synthesized at the same UDP-sugar concentrations. The model indicates that translocation occurs following the addition of GlcUA to the chain terminus, whereas UDP-Glc drives chain termination when inadequate levels of UDP-GlcUA are present. In sum, type 3 synthase appears to modulate polysaccharide chain length by functioning as a concentration-dependent kinetic timing device.

The processive reaction mechanisms of beta-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of beta-1,3-Glc and beta-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 mum, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.

Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited gamma-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed beta-d-N-acetylglucosaminidase activity.

Regulation of chain length is essential to the proper functioning of prokaryotic and eukaryotic polysaccharides. Modulation of polymer size by substrate concentration is an attractive but unexplored control mechanism that has been suggested for many polysaccharides. The Streptococcus pneumoniae capsular polysaccharide is essential for virulence, and regulation of its size is critical for survival in different host environments. Synthesis of the type 3 capsule [-4)-beta-d-Glc-(1-3)-beta-d-GlcUA-(1-] from UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) is catalysed by the type 3 synthase, a processive beta-glycosyltransferase, and requires a UDP-Glc dehydrogenase for conversion of UDP-Glc to UDP-GlcUA. Strains containing mutant UDP-Glc dehydrogenases exhibited reduced levels of UDP-GlcUA, along with reductions in total capsule amount and polymer chain length. In both the parent and mutant strains, UDP-Glc levels far exceeded UDP-GlcUA levels, which were very low to undetectable in the absence of blocking synthase activity. The in vivo observations were consistent with in vitro conditions that effect chain termination and ejection of the polysaccharide from the synthase when one substrate is limiting. These data are the first to demonstrate modulation of polysaccharide chain length by substrate concentration and to enable a model for the underlying mechanism. Further, they may have implications for the control of chain length in both prokaryotic and eukaryotic polymers synthesized by similar mechanisms.

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.

The type 3 synthase from Streptococcus pneumoniae is a processive beta-glycosyltransferase that assembles the type 3 polysaccharide [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->] by a multicatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nucleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-[(14)C]Glc or UDP-[(14)C]GlcUA, we found that phospholipase D digestion of the Glc-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Glc followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not covalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D approximately P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rx1, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D approximately P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rx1 background, is important in the control of capsule synthesis.

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.

The type 3 polysaccharide synthase from Streptococcus pneumoniae catalyzes sugar transfer from UDP-Glc and UDP-glucuronic acid (GlcUA) to a polymer with the repeating disaccharide unit of [3)-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->]. Evidence is presented that release of the polysaccharide chains from S. pneumoniae membranes is time-, temperature-, and pH-dependent and saturable with respect to specific catalytic metabolites. In these studies, the membrane-bound synthase was shown to catalyze a rapid release of enzyme-bound polysaccharide when either UDP-Glc or UDP-GlcUA alone was present in the reaction. Only a slow release of polysaccharide occurred when both UDP sugars were present or when both UDP sugars were absent. Chain size was not a specific determinant in polymer release. The release reaction was saturable with increasing concentrations of UDP-Glc or UDP-GlcUA, with respective apparent K(m) values of 880 and 0.004 micrometer. The apparent V(max) was 48-fold greater with UDP-Glc compared with UDP-GlcUA. The UDP-Glc-actuated reaction was inhibited by UDP-GlcUA with an approximate K(i) of 2 micrometer, and UDP-GlcUA-actuated release was inhibited by UDP-Glc with an approximate K(i) of 5 micrometer. In conjunction with kinetic data regarding the polymerization reaction, these data indicate that UDP-Glc and UDP-GlcUA bind to the same synthase sites in both the biosynthetic reaction and the chain release reaction and that polymer release is catalyzed when one binding site is filled and the concentration of the conjugate UDP-precursor is insufficient to fill the other binding site. The approximate energy of activation values of the biosynthetic and release reactions indicate that release of the polysaccharide occurs by an abortive translocation process. These results are the first to demonstrate a specific enzymatic mechanism for the termination and release of a polysaccharide.

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.