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R W Le Page

Publications and source records attributed to R W Le Page.

At least 19 recordsLinked to original sources
PubMed2000

Heterologous expression of an immunogenic pneumococcal type 3 capsular polysaccharide in Lactococcus lactis.

In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogen Streptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter(-1)) of an extracellular type 3 CPS in L. lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L. lactis. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L. lactis and S. pneumoniae showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 x 10(6) CFU of live recombinant lactococci expressing a total of approximately 0.5 microgram of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 microgram of type 3 CPS purified from S. pneumoniae. These findings show that L. lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally, L. lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.

Animals↗
PubMed1999

6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme.

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1. 44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4+/-1.4 microM and for NADP of 1.9+/-0.2 microM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2'-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.

Amino Acid Sequence↗
PubMed1998

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine.

Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains of L. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains of L. lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.

Administration, Intranasal↗
PubMed1997

Oral vaccination of mice against tetanus with recombinant Lactococcus lactis.

To determine whether a protective immune response could be elicited by oral delivery of a recombinant bacterial vaccine, tetanus toxin fragment C (TTFC) was expressed constitutively in Lactococcus lactis and administered orally to C57 BL/6 mice. The antibody titers elicited were lower than those following intranasal immunization (a route already known to result in high-level systemic anti-TTFC immune responses) but the protective efficacy was the same order of magnitude. The serum antibody isotypes elicited were predominantly IgG1 and IgG2a. TTFC-specific fecal IgA responses could be detected following oral or intranasal immunization. Chemically killed lactococci administered via the intranasal route were also able to elicit serum antibody responses of similar levels and kinetics to those induced by live bacteria.

Administration, Intranasal↗
PubMed1996

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis.

The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.

Animals↗
PubMed1996

An origin of DNA replication from Lactococcus lactis bacteriophage c2.

An origin of DNA relication was identified in the intergenic region between the early and late gene regions of prolate lactococcal phage c2. A DNA fragment containing this origin, designated ori, was shown to direct DNA replication in Lactococcus lactis but not in Escherichia coli. A comparison of ori with the corresponding regions of other prolate phages revealed strict conservation of the nucleotide sequence in one half of this intergenic region. This conserved region alone would not support DNA replication. No open reading frames were identified in the ori fragment, suggesting that host factors alone are sufficient to initiate DNA replication at ori. A novel class of lactococcal vectors and E. coli-L. lactis shuttle vectors based on ori have been constructed.

Base Sequence↗
PubMed1995

The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis.

18 different promoter elements, encompassing a 71-fold range of activity, were isolated from the chromosome of Lactococcus lactis (Ll) MG1363 and from an uncharacterised small isometric bacteriophage of Ll. The Vibrio fischeri (Vf) luciferase-encoding gene (lux) was used as a reporter in Ll, so that the promoters could be identified strictly on the basis of their activity in the homologous host. Sequence and primer extension analysis of six of the promoters has provided a new consensus sequence for the -35 and -10 hexanucleotide motifs present upstream from lactococcal transcription start points. When the nucleotide sequence of the most active promoter (P15) was compared with that of the highly expressed Ll usp45 gene, a novel 8-bp region of homology was identified which corresponded to the newly derived consensus -35 sequence element; this element may therefore be of general importance in Ll gene expression. The isolation of these promoters has also enabled us to investigate the characteristics of the Vf Lux activity in Ll under different physiological conditions using promoters of different strengths. Lux activity in Ll is critically dependent upon the phase of cell growth. Luminescence falls sharply in stationary phase, possibly due to a lack of FMNH2. In contrast to the kinetics of Lux function in Escherichia coli (Ec), Lux activity in Ll declines rapidly after addition of the substrate; the rate of decay is dependent both on the growth phase and on the strength of the promoter. It is apparent that the previously reported thermal instability of Lux is in fact a function of the host organism in which Lux is expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Progress in the development of Lactococcus lactis as a recombinant mucosal vaccine delivery system.

The non-pathogenic, non-colonising Gram-positive organism Lactobacillus lactis is beeing developed as an antigen delivery system for mucosal vaccination. A high level expression system has been developed which allows loading of the bacterium with high levels of a heterologous antigen (TTFC) prior to inoculation. Mucosal inoculation of one such recombinant strain results in a protective serum antibody response and production of TTFC-specific IgA at mucosal sites.

Animals↗
PubMed1995

Sequencing and analysis of the prolate-headed lactococcal bacteriophage c2 genome and identification of the structural genes.

The 22,163-bp genome of the lactococcal prolate-headed phage c2 was sequenced. Thirty-nine open reading frames (ORFs), early and late promoters, and a putative transcription terminator were identified. Twenty-two ORFs were in the early gene region, and 17 were in the late gene region. Putative genes for a DNA polymerase, a recombination protein, a sigma factor protein, a transcription regulatory protein, holin proteins, and a terminase were identified. Transcription of the early and late genes proceeded divergently from a noncoding 611-bp region. A 521-bp fragment contained within the 611-bp intergenic region could act as an origin of replication in Lactococcus lactis. Three major structural proteins, with sizes of 175, 90, and 29 kDa, and eight minor proteins, with sizes of 143, 82, 66, 60, 44, 42, 32, and 28 kDa, were identified. Several of these proteins appeared to be posttranslationally modified by proteolytic cleavage. The 175- and 90-kDa proteins were identified as the major phage head proteins, and the 29- and 60-kDa proteins were identified as the major tail protein and (possibly) the tail adsorption protein, respectively. The head proteins appeared to be covalently linked multimers of the same 30-kDa gene product. Phage c2 and prolate-headed lactococcal phage bIL67 (C. Schouler, S. D. Ehrlich, and M.-C. Chopin, Microbiology 140:3061-3069, 1994) shared 80% nucleotide sequence identity. However, several DNA deletions or insertions which corresponded to the loss or acquisition of specific ORFs, respectively, were noted. The identification of direct nucleotide repeats flanking these sequences indicated that recombination may be important in the evolution of these phages.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The immune response to Lactococcus lactis: implications for its use as a vaccine delivery vehicle.

The development of natural antibodies to Lactococcus lactis in three inbred strains of mice and the effect of inoculating L. lactis into these mice has been investigated. All animals developed detectable levels of natural (systemic and secretory) anti-lactococcal antibodies. Systemic anti-lactococcal antibodies were principally IgG in young mice. An increase in anti-lactococcal IgM occurred in all older animals. Inoculation of L. lactis strains MG1820 or MG1363 by the oral and parenteral routes induced systemic anti-lactococcal antibody responses in a dose-dependent fashion. The relative immunogenicity of individual bacterial proteins varied according to mouse strain and route of inoculation.

Animals↗
PubMed1994

Overexpression in Escherichia coli and purification of the 6-phosphogluconate dehydrogenase of Trypanosoma brucei.

The gene encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Trypanosoma brucei was cloned into the overexpression vector pET3a which utilizes the T7 polymerase gene expression system. Up to 40% of total cell protein consisted of the trypanosome enzyme when expression was induced in Escherichia coli host strains at 28 degrees C. The enzyme was rapidly degraded at temperatures higher than 30 degrees C. The T. brucei enzyme was purified to near homogeneity (as judged by SDS-polyacrylamide gel electrophoresis) using a two-step purification method, involving a DE-52 cellulose batch preparation followed by 2' AMP-agarose affinity chromatography. The purified protein crystallized readily. A molecular model of the trypanosome enzyme based on its mammalian counterpart revealed differences between the two enzymes in residues involved in cofactor binding.

Animals↗
PubMed1993

Preliminary crystallographic study of 6-phosphogluconate dehydrogenase from Trypanosoma brucei.

We have obtained well-ordered single crystals of 6-phosphogluconate dehydrogenase, an enzyme of the oxidative branch of the pentose phosphate pathway, from Trypanosoma brucei. The crystals are trigonal rhombs with unit cell dimensions a = b = 135.1 A, c = 116.7 A and belong to one of the enantiomorphic pair of space groups P3(1)21/P3(2)21. X-ray diffraction to better than 2.8 A has been recorded using a rotating anode CuK alpha source. Elucidation of the three-dimensional structure of the T. brucei enzyme will, by indicating structural differences from the known sheep enzyme structure, aid the design of mutants to probe the active site. Knowledge of the structure will also assist in assessing the potential use of rationally designed compounds to inhibit this enzyme specifically.

Animals↗
PubMed1993

A 6-phosphogluconate dehydrogenase gene from Trypanosoma brucei.

A Trypanosoma brucei gene encoding 6-phosphogluconate dehydrogenase (6-PGDH) (EC 1.1.1.44) was identified and cloned by functional complementation of Escherichia coli gnd mutants with genomic trypanosome DNA. The T. brucei gnd gene is present as a single copy. In Northern blot experiments a probe derived from the gene hybridises to 2 transcripts (2.9 kb and 3.1 kb) which are found in both bloodstream and procyclic form organisms; the larger transcript is more abundant in bloodstream form organisms. The derived amino acid sequence of the protein is 479 amino acids in length, with a molecular weight of 52,000. It is homologous to 6-PGDHs from bacterial and mammalian sources, but diverges significantly from these other enzymes.

Amino Acid Sequence↗
PubMed1993

Improved cloning vectors and transformation procedure for Lactococcus lactis.

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L. lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v. Although growth of L. lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.

Base Sequence↗