Key publications | Publications by category | Publications by year

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.

Galleria mellonella (wax moth) larvae have elsewhere been shown to be susceptible to pathogens such as Francisella tularensis, Burkholderia mallei, and Pseudomonas aeruginosa. We report that the larvae are rapidly killed by Campylobacter jejuni at 37C. Three strains of C. jejuni tested, 11168H (human diarrheal isolate), G1 (human Guillain-Barré syndrome isolate), and 81-176 (human diarrheal isolate), were equally effective at killing G. mellonella larvae. A panel of defined mutants of C. jejuni 11168H, in known or putative virulence genes, showed different degrees of attenuation in G. mellonella larvae. A mutant lacking the O-methyl phosphoramidate (MeOPN) capsule side group was attenuated, clearly demonstrating that MeOPN has a role in virulence. This new model of C. jejuni infection should facilitate the identification of novel virulence genes.

BACKGROUND: We aimed to determine the antibody and T cell responses to Burkholderia pseudomallei of humans to select candidate vaccine antigens. METHODS: for antibody profiling, a protein microarray of 154 B. pseudomallei proteins was probed with plasma from 108 healthy individuals and 72 recovered patients. Blood from 20 of the healthy and 30 of the recovered individuals was also obtained for T cell assays. RESULTS: Twenty-seven proteins distinctively reacted with human plasma following environmental exposure or clinical melioidosis. We compared the responses according to the patient's history of subsequent relapse, and antibody response to BPSL2765 was higher in plasma from individuals who had only 1 episode of disease than in those with recurrent melioidosis. A comparison of antibody and T cell responses to 5 B. pseudomallei proteins revealed that BimA and flagellin-induced responses were similar but that BPSS0530 could induce T cell responses in healthy controls more than in recovered patients. CONCLUSIONS: By combining large-scale antibody microarrays and assays of T cell-mediated immunity, we identified a panel of novel B. pseudomallei proteins that show distinct patterns of reactivity in different stages of human melioidosis. These proteins may be useful candidates for development of subunit-based vaccines and in monitoring the risks of treatment failure and relapse.

(1996). Role of two outer membrane antigens in the induction of protective immunity against Francisella tularensis strains of different virulence.

Abstract: Role of two outer membrane antigens in the induction of protective immunity against Francisella tularensis strains of different virulence.

Duffield, M. (2006). Comparative genomics – what do such studies tell us about the emergence and spread of. key pathogens. In Logan NA, Lappin-Scott HM, Oyston PCF (Eds.) Prokaryotic diversity: mechanisms and significance, Cambridge University Press, 175-186.

An extracellular haemolytic activity, produced by the fish pathogen Aeromonas salmonicida, against trout erythrocytes (T-lysin) was partially purified by ammonium sulphate precipitation and gel chromatography. Lysis of trout erythrocytes was found to be due to the combined activity of a caseinase and another factor, apparently membrane-associated, which when separated caused incomplete lysis. Incomplete lysis was also observed when caseinase production was suppressed by the incorporation of ammonium sulphate in the growth medium, or in caseinase-negative mutants. Inhibition of caseinase activity by phenyl methyl sulphonyl fluoride also resulted in the loss of full lytic potential from culture supernates containing T-lysin. © 1983.

H-lysin from Aeromonas salmonicida has been purified 1770-fold by freeze fractionation, ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The purified material was predominantly H-lysin, devoid of detectable T-lysin, caseinase or gelatinase activity, although glycerophospholipid: cholesterol acyltransferase (GCAT) activity was present. The results suggested that H-lysin and GCAT activities were due to different extracellular products. Studies of the kinetics of haemolysis indicated that the H-lysin had an enzymic mode of action, and that initial erythrocyte damage appeared to precede lysis of the cell. The H-lysin was lethal to cultured rainbow trout gonad cells and leucocytes, but when it was injected intravenously in rainbow trout no pathological effects were observed.

Mutants of the bacterial fish pathogen Aeromonas salmonicida selected for inability to digest casein concomitantly lost hemolytic activity against horse erythrocytes under certain conditions. Mixtures of wild-type with mutant culture supernatants indicated that mutants produce an inactive precursor of a hemolysin which was activated by autogenous caseinase and, with less efficiency, by other serine proteases. Selective inhibition or repression of caseinase production in the wild-type strain also resulted in the production of an inactive precursor of a hemolysin. The precursor of hemolysin was also activated by a serum factor which appeared to exert its maximum effect at the bacterial surface or after entry into the bacterial cell. These results could affect the interpretation of studies evaluating the role of individual extracellular products in the pathogenesis of A. salmonicida infections.

Spores of Bacillus anthracis germinated poorly at high cell densities unless the alanine racemase inhibitor O-carbamyl-D-serine was added to the germination medium. Spores derived from a variety of strains of B. anthracis germinated optimally at 22 degrees C. No correlation was found between rate of spore germination and virulence or between susceptibility of animal species to anthrax and spore germination rate using sera from those animals as the germination medium.

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.

(1991). Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway. Infect Immun, 59(5), 1872-1874.

The N-terminal domain of Clostridium perfringens alpha-toxin, homologous with the nontoxic phospholipase C of Bacillus cereus, was expressed in Escherichia coli and shown to retain all of the phosphatidylcholine hydrolyzing activity of the alpha-toxin, but not the sphingomyelinase, hemolytic, or lethal activities. The C-terminal domain of alpha-toxin showed sequence and predicted structural homologies with the N-terminal region of arachidonate 5-lipoxygenase, an enzyme from the human arachidonic acid pathway which plays a role in inflammatory and cardiovascular diseases in humans.

Abstract: Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway.

A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.

The polymerase chain reaction (PCR) was used to identify spores of Bacillus anthracis. By using an assay capable of amplifying a 1247-bp fragment from the gene that encodes the edema factor of B. anthracis, as few as 10(3) copies of a plasmid containing the edema factor gene and as few as 2 x 10(4) spores were detected. Subjecting the product of this PCR to a second PCR designed to amplify a 208-bp fragment nested within the 1247-bp product improved detection to a single plasmid copy per PCR and to two spores of B. anthracis per PCR.

(1992). Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D. FEMS Microbiol Lett, 76(1-2), 77-81.

The sequence of the epsilon toxin gene of Clostridium perfringens type D was determined and compared with that of the previously reported type B sequence. It showed two nucleotide changes in the open reading frame, giving rise to one amino acid substitution. The promoter sequences were not homologous, and different putative -35 and -10 regions have been identified in each. The sequence information was used to develop PCR primers which were specific for the epsilon toxin gene. The utility of this system for identifying type B or D strains of C. perfringens was demonstrated.

Abstract: Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D.

The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin.

Two conserved regions in the sequence of the NS5 gene of Flaviviruses were identified. Primers were designed from the consensus sequence of these regions and were used in a reverse transcription/polymerase chain reaction (RT/PCR) to amplify a region of the central european tick-borne encephalitis virus Kumlinge NS5 gene. The authenticity of the amplified fragment was confirmed by nucleotide sequencing. A band of the expected size was also obtained when this RT/PCR was applied to 13 other flaviviral RNAs. This method may be useful for characterisation of the NS5 genes of flaviviruses and as a potential pan-flavivirus diagnostic tool.

Oligonucleotide probes designed on the basis of the N-terminal sequence of Clostridium perfringens beta-toxin were used to isolate the encoding gene (cpb). The nucleotide sequence of cpb was determined, and on the basis of DNA hybridization experiments it was shown that the gene is found only in type B and C strains of C. perfringens. The deduced amino acid sequence of the beta-toxin revealed homology with the alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. The beta-toxin purified from C. perfringens appeared to exist in monomeric and multimeric forms. Recombinant beta-toxin, produced in Escherichia coli, appeared to be mainly in the multimeric form.

The C-terminal domain of the alpha-toxin (cpa247-370) of Clostridium perfringens has been expressed in Escherichia coli and purified. Antiserum raised against cpa247-370 reacted in an identical manner to anti-alpha-toxin serum when used to map epitopes in the C-terminal domain, suggesting that cpa247-370 was immunologically and structurally identical to this region in the alpha-toxin. The isolated cpa247-370 was devoid of sphingomyelinase activity or haemolytic activity and was not cytotoxic for mouse lymphocytes. Haemolytic activity was detected when cpa247-370 was tested with the N-terminal domain of the alpha-toxin (cpa1-249), confirming that cpa247-370 confers haemolytic properties on the phospholipase C activity of the alpha-toxin. Haemolytic activity was not detected if cpa247-370 was tested with the Bacillus cereus phosphatidylcholine phospholipase C, nor if cpa1-249 and cpa247-370 were incubated sequentially with erythrocytes.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.

We have determined the nucleotide sequence of fopA from Francisella tularensis. Using the polymerase chain reaction fopA was detected in high and low virulence biotypes of F. tularensis. fopA was stably maintained in pBluescript in attenuated Salmonella typhimurium where FopA was expressed and located in the outer membrane. This recombinant will be suitable for studies on the role of FopA in immunity against tularaemia.

Fragments of the alpha-toxin of Clostridium perfringens have been produced using genetic manipulation techniques. Antibody which cross-reacted with the alpha-toxin was induced after immunization with fragments representing the N- (Cpa1-249) and C-terminal (Cpa247-370) domains of the toxin. Smaller fragments of the alpha-toxin did not induce cross-reacting antibody. Anti-Cpa1-249 serum neutralized phospholipase C activity but not haemolytic activity of the toxin. Anti-Cpa247-370 serum neutralized both the phospholipase C and haemolytic activities. Only immunization with Cpa247-370 induced protection against the lethal effects of the toxin. Immunization with Cpa247-370 also provided protection in a mouse model against at least 10 LD100 doses of C. perfringens type A. This result confirms the essential role of this toxin in the pathogenesis of gas gangrene.

Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C. The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

Polymerase chain reactions (PCRs) for the capsule and oedema factor genes of Bacillus anthracis were used to assess methods for detecting B. anthracis spores. Untreated spore preparations were found to contain significant amounts of extracellular template DNA which probably accounted for observed amplification from these preparations without spore lysis. Germination of spores with suitable media allowed the detection of less than 10 spores in a PCR test. Mechanical disruption of spores with glass or zirconia beads yielded similar results to germination but in a much shorter time. The techniques described should improve the detection by PCR of B. anthracis and other sporulating bacteria. Copyright © 1994, Wiley Blackwell. All rights reserved

We have used the technique of antibody reshaping to produce a humanized antibody specific for the alpha toxin of Clostridium perfringens. The starting antibody was from a mouse hybridoma from which variable (V) region nucleotide sequences were determined. The complementarity-determining regions (CDRs) from these V regions were then inserted into human heavy and light chain V region genes with human constant region gene fragments subsequently added. The insertion of CDRs alone into human frameworks did not produce a functional reshaped antibody and modifications to the V region framework were required. With minor framework modifications, the affinity of the original murine mAb was restored and even exceeded. Where affinity was increased, an altered binding profile to overlapping peptides was observed. Computer modelling of the reshaped heavy chain V regions suggested that amino acids adjacent to CDRs can either contribute to, or distort, CDR loop conformation and must be adjusted to achieve high binding affinity.

The stability of plasmids pBR322, pUC19 and pBluescript and their effect on bacterial colonization of mice was determined. S. typhimurium SL 3261 carrying high copy number plasmids colonized spleen and liver tissues poorly compared to low copy number plasmids. After inoculation into mice, the stability of the plasmids appeared to be inversely related to the plasmid's size and complexity. Mouse-passaging a pBluescript-based recombinant plasmid expressing the Protective Antigen of Bacillus anthracis selected for a mutant S. typhimurium strain (designated G3) that colonized at high levels and more stably maintained plasmids than S. typhimurium SL 3261. S. typhimurium G3 down-regulated the copy number of ColE1 plasmids. The significance of these data for vaccine design is discussed.

The protective antigen (PA) gene from Bacillus anthracis has been expressed in Salmonella typhimurium SL 3261 (aroA). Expression was achieved by cloning the gene after the plac promoter in a high copy number plasmid. The recombinant PA was exported into the periplasm. This construct was unstable in vivo and also reduced the colonization ability of the host S. typhimurium. Mouse-passaging of the recombinant Salmonella resulted in a strain with enhanced colonization ability and increased stability of the plasmid in vivo. This effect appeared to be due to a reduction in copy number of the PA-encoding plasmid. Mice were vaccinated with recombinant S. typhimurium and adjuvanted PA and challenged with virulent B. anthracis. Only mice vaccinated with adjuvanted PA or orally with the mouse-passaged recombinant showed partial protection. The degree of protection observed after oral vaccination with the recombinant S. typhimurium was similar to the degree of protection afforded by adjuvanted PA and suggested that the use of S. typhimurium to deliver PA is an effective approach for inducing protection against B. anthracis. The results presented also suggest that the degree of protection demonstrated in the mouse may not fully indicate the potential of the recombinant Salmonella as an effective vaccine in other species.

A reporter system was constructed to enable the study of gene expression in Clostridium perfingens. The system was based on plasmid shuttle vector pJIR410, which contained the C. perfringens erythromycin resistance gene. The vector was modified by the introduction of a DNA fragment comprising the open reading frame of the C. perfringens chloramphenicol acetyltransferase gene and flanking transcriptional terminators. The presence of a unique restriction site, engineered into the extreme 5' end of the open reading frame enabled a promoter region to be inserted to form an in-fram transcriptional fusion with catP. The system was tested by inserting the promoter region of the alpha-toxin gene of C. perfringens. The production of chloramphenicol acetyltransferase in C. perfringens was monitored during growth and the pattern of expression was shown to reflect levels of plc mRNA and alpha-toxin in the parent strain.

A crude outer membrane preparation from Francisella tularensis Live Vaccine Strain (LVS) was used to immunize mice. Immunized mice were completely protected from a F. tularensis challenge. We evaluated the role of two major outer membrane antigens in the induction of protective immunity, namely lipopolysaccharide and an outer membrane protein FopA. We presented FopA to the immune system using an aromatic amino acid-dependent Salmonella typhimurium as a vector. Although mice mounted an immune response to cloned FopA no significant protection was induced. However, LPS immunized mice were completely protected. We conclude that LPS is a major protective antigen whereas FopA has a limited or no role in the induction of protective immunity.

Banzi is a mosquito borne flavivirus which belongs to the Uganda S serocomplex. No nucleotide sequence data have previously been reported from any virus of this serocomplex. We have determined the nucleotide sequence of the NS5 gene from Banzi virus and the predicted amino acid sequence was elucidated. Previously identified conserved RNA polymerase, methyltransferase and flavivirus NS5 amino acid motifs were present in the Banzi virus NS5 protein. These data add to the evidence for the functional importance of the regions. The encoded amino acid sequence was compared with the predicted amino acid sequence of other flavivirus NS5 proteins. Analysis of these sequences suggested that Banzi virus is most closely related to the mosquito-borne flaviviruses and, in particular, yellow fever virus. This pattern of similarity is in accordance with the previously suggested serological classification of flaviviruses.

An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.

In this study, we have determined the limit of protection achievable by immunisation with sub-units of Yersinia pestis against the development of plague in an experimental animal model. Co-immunisation with the purified culture-derived F1 and the recombinant V sub-units afforded a greater level of protection than with either sub-unit alone. The protection given by the combined sub-units was several orders of magnitude greater than that afforded by the whole cell killed (Cutter USP) vaccine and was equivalent to that achieved by vaccination with EV76, the live attenuated Y. pestis vaccine strain. However, the combined sub-unit vaccine has clear advantages over the live vaccine in terms of safety of use and absence of side-effects. © 1995.

The median lethal dose (MLD) of a pathogenic strain of Yersinia pestis was established by three routes of administration in three strains of mouse. There was no significant difference between the MLDs in the different strains of mouse. The MLD by the subcutaneous route in Balb/C and an outbred line was approximately 1 c.f.u.; the MLD following intraperitoneal administration was tenfold higher. There were significant differences in the mean times to death after administration of the challenge by different routes. The relative efficacy of a live attenuated vaccine strain of Y. pestis (EV76) was compared with that of the formaldehyde-killed vaccine (Plague vaccine, USP). EV76 protected against high challenge doses (up to 5.75 x 10(6) MLD), though immunized animals showed side effects of varying severity. The killed vaccine was less effective in terms of dose-protection (deaths occurred after challenge with 4000 MLD) and several of the vaccinated animals suffered sub-lethal, plague-related sequelae to the challenge.

Conditions were defined for the successful transformation of the human pathogen Burkholderia pseudomallei 4845 by electroporation, using the incQ plasmid pKT230. We have obtained frequencies of up to 8 x 10(3) transformants per microgram plasmid DNA with freshly prepared electrocompetent B. pseudomallei. The method also allows for easy and reproducible production of frozen cell suspensions which can produce efficiencies up to 2.5 x 10(2) transformants per microgram of plasmid DNA. Kanamycin had to be added to the culture growth medium at a minimum concentration of 20 micrograms ml-1 and a maximum concentration of 50 micrograms ml-1 for the bacteria to become electrocompetent. Bacteria grown in medium containing a final concentration of 30 micrograms ml-1 kanamycin produced the highest numbers of transformants, although the transformation frequency at this concentration was not as efficient as that at 50 micrograms ml-1. Electron microscopy indicated that the kanamycin affects the integrity of the bacterial outer membrane, which becomes loose and wrinkled in appearance. The electrocompetence does not result in a permanent morphological change.

(1996). The level of expression of α-toxin by different strains of Clostridium perfringens is dependent on differences in promoter structure and genetic background. Anaerobe, 2(6), 365-371.

The control of expression of the α-toxin gene (cpa or plc) of Clostridium perfringens has been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related to cpa mRNA levels. Primer extension studies were performed to locate the cpa promoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237 cpa gene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.

Abstract: The level of expression of α-toxin by different strains of Clostridium perfringens is dependent on differences in promoter structure and genetic background

The effect of buffer composition on simultaneous PCR amplification of 16S rRNA gene fragments of five bacterial species was examined using a number of different buffer systems. Tris-based PCR buffers at final concentrations of 10 mM proved unreliable. However, when the final concentration of Tris was increased to 75 mM, all five samples were routinely detected. The use of other buffers, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO) and 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO), resulted in PCR amplification of five products even at low final concentrations (10 mM). The presence of certain proteins in the amplification reaction could overcome an inhibitory effect seen when soil suspension was present in the reaction, as might occur when testing field samples for the presence of bacteria. Bovine serum albumin was found to be the most effective additive tested in overcoming inhibition.

The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.

(1996). Molecular variation between the alpha-toxins from the type strain (NCTC 8237) and clinical isolates of Clostridium perfringens associated with disease in man and animals. Microbiology (Reading), 142 ( Pt 1), 191-198.

The alpha-toxin produced by the type strain of Clostridium perfringens (NCTC 8237) was shown to differ from the alpha-toxins produced by most strains of C. perfringens isolated from man and from calves with respect to reactivity with a neutralizing monoclonal antibody (DY2F5D11). The difference in antibody binding correlated with three differences in the deduced amino acid sequence (Ala174 to Asp174; Thr177 to Ala177; Ser335 to Pro335) of the alpha-toxins. Using octapeptides synthesized on the basis of the amino acid sequences from these regions of variability, it was shown that the Ala174 to Asp174 change had the greatest effect on reducing the binding of monoclonal antibody DY2F5D11 to the alpha-toxin. These differences did not affect the enzymic or toxic properties of the protein. However, the phospholipase C activity of the alpha-toxin produced by strain NCTC 8237 was more susceptible to inactivation by chymotrypsin. The changes in amino acid sequence did not affect the ability of a C-terminal domain vaccine, derived from the alpha-toxin of strain NCTC 8237, to induce protection against the alpha-toxin from a bovine enteric strain of C. perfringens.

Abstract: Molecular variation between the alpha-toxins from the type strain (NCTC 8237) and clinical isolates of Clostridium perfringens associated with disease in man and animals.

Microencapsulated Fl and V sub-unit antigens of Yersinia pestis were used to immunize mice intraperitoneally with a combination of 25 micrograms of each of the microencapsulated sub-units. The combined microsphere formulation induced both mucosal and systemic immunity. There was an additive effect in combining sub-units and the protection afforded by the combined microencapsulated antigens was superior to that provided by the administration of any single encapsulated antigen and by the existing whole cell vaccine. The protective efficacy of the combined microencapsulated sub-units was further enhanced by co-administering cholera toxin B sub-unit. Microencapsulation of the sub-units offered advantages which included depot release of the vaccine in vivo and the facilitation of oral, intranasal or inhalational delivery. Therefore, immunization with microencapsulated sub-unit antigens was an effective means of generating humoral and cellular responses which endowed protective immunity.

The efficacy of doxycycline and ciprofloxacin against an experimental plague infection was assessed by comparing the median lethal dose (MLD) of Yersinia pestis in antibiotic-treated and untreated mice. The MLD of Y. pestis GB strain in untreated mice by the intra-peritoneal route was 23 cfu. If ciprofloxacin dosage (20 or 40 mg/kg twice daily) was initiated 48 h before infection, it afforded complete protection against an intra-peritoneal challenge of 5.24 x 10(7) cfu. Ciprofloxacin therapy initiated 24 h post-challenge was less protective, the MLD was raised to 2.0 x 10(5) and 2.2 x 10(5) cfu for 40 and 20 mg/kg respectively. Doxycycline dosage (40 mg/kg twice daily) initiated 48 h prior to infection raised the MLD to 1.6 x 10(4) cfu, but other prophylactic and therapeutic regimes were ineffective against challenges greater than 6.76 x 10(2) cfu. Ciprofloxacin may therefore be a useful antibiotic to consider for the treatment of plague.

Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.

This study describes a PCR-based approach for the production of a rationally attenuated mutant of Yersinia pestis. Degenerate primers were used to amplify a fragment encoding 91.45% of the aroA gene of Y. pestis MP6 which was cloned into pUC18. The remainder of the gene was isolated by inverse PCR. The gene was sequenced and a restriction map was generated. The Y. pestis aroA gene had 75.9% identity with the aroA gene of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain GB using the suicide vector pGP704. A stable aro-defective mutant. Y. pestis GB aroA, was isolated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response against challenge with the virulent Y. pestis strain GB. Unusually for an aro-defective mutant, the Y. pestis aroA mutant was virulent in mice, with a median dose which induced morbidity of death similar to that of the wild-type, although time to death was significantly prolonged.

We have developed a highly sensitive method for detection of Francisella tularensis in clinical samples based on a nested polymerase chain reaction (PCR) for the FopA gene. Mice infected with F. tularensis were killed at 24-hr intervals, and the DNA from blood and spleens was extracted by a variety of methods and analyzed by PCR. The best method, based on the ability of DNA to bind to silica in the presence of guanidine thiocyanate, yielded amplifiable DNA without dilution of the murine tissue samples. Francisella tularensis in infected murine spleens and culture-positive blood samples was reliably detected by nested PCR following this extraction procedure. We believe this technique has significant advantages over traditional methods for diagnosing F. tularensis infection in terms of speed, ease of use, reproducibility, and safety.

V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. A series of V-antigen truncates expressed as glutathione S-transferase (GST) fusion proteins (GST-V truncates) have been cloned and purified to support immunogenicity and functionality studies of V antigen. Immunization studies with GST-V truncates have identified two regions of V antigen that confer protection against Y. pestis 9B (a fully virulent human pneumonic plague isolate) in a mouse model for plague. A minor protective region is located from amino acids 2 to 135 (region I), and a major protective region is found between amino acids 135 and 275 (region II). In addition, analysis of IgG titers following immunization suggested that the major antigenic region of V antigen is located between amino acids 135 and 245. A panel of monoclonal antibodies raised against recombinant V antigen was characterized by Western blotting against GST-V truncates, and epitopes of most of the monoclonal antibodies were mapped to region I or II. Monoclonal antibody 7.3, which recognizes an epitope in region II, passively protected mice against challenge with 12 median lethal doses of Y. pestis GB, indicating that region II encodes a protective epitope. This is the first report of a V-antigen-specific monoclonal antibody that will protect mice against a fully virulent strain of Y. pestis. The combined approach of passive and active immunization has therefore confirmed the importance of the central region of the protein for protection and also identified a previously unknown protective region at the N terminus of V antigen.

In response to an outbreak of a plague-like disease in India, the Public Health Laboratory Service (PHLS) in the UK distributed advice on the isolation and identification of Yersinia pestis. Some of the procedures outlined were evaluated using a number of isolates of Y. pestis, complemented with in-house techniques detecting virulence genes or their products. These laboratory investigations are limited in that they are either only indicative or they take too long (48 hours or more), and thus represent a serious delay to the patient. Successful patient management must be based on a case history, and therapy should be started immediately. Laboratory diagnosis will subsequently rule out most pathogens which cause similar infections, yet will still require confirmation by a reference laboratory.

(1997). Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague. Infect Immun, 65(5), 1926-1930.

The caf operon from Yersinia pestis encoding the structural subunit (caf1), the molecular chaperone (caf1M), the outer membrane anchor (caf1A), and the regulatory protein (caf1R) was cloned into Salmonella typhimurium SL3261 aroA. The recombinant Salmonella organisms were encapsulated when cultured at 37 degrees C but not when cultured at 28 degrees C. Oral inoculation of mice with the recombinant Salmonella induced predominantly an immunoglobulin G2a response to F1 antigen, and isolated T cells showed a recall response to soluble or Salmonella-associated F1 antigen. Mice immunized with S. typhimurium SL3261 aroA expressing F1 antigen intracellularly developed lower antibody responses to F1 antigen and showed a T-cell recall response only to Salmonella-associated F1 antigen. Mice immunized orally with two doses of the recombinant Salmonella which expressed F1 antigen on the surface were protected against 10(7) 50% lethal doses (LD50) of virulent Y. pestis given by the subcutaneous route of challenge, whereas mice immunized with the recombinant Salmonella expressing F1 antigen intracellularly were only partially protected against 10(5) LD50 of Y. pestis.

Abstract: Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague.

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed. The nucleotide sequences encoding Yersinia pestis V antigen (lcrV) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3' end of caf1 and the 5' end of lcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuated Salmonella typhimurium strain SL3261 (aroA). Mice receiving two intravenous doses of 5 x 10(6) cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG1:IgG2a isotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 x 10(2) or 7.4 x 10(4) LD50s of Y. pestis strain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressing Y. pestis antigens as fusion proteins to develop a live recombinant vaccine against plague.

In this study, the protection afforded against aerosolized Yersinia pestis by injection of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine protected mice against exposure to > 10(4) colony-forming units (c.f.u.) of virulent plague organisms (100 LD50 doses), whereas the whole cell vaccine provided only 50% protection against 1.8 x 10(3) c.f.u. In sub-unit vaccinees, IgG to each of the F1 and V antigens contained in the vaccine, was detected in serum, on direct secretion by spleen cells and in broncho-alveolar washings (BAL). In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable in equivalent samples. Levels of F1-specific IgG in serum, secreted from spleen cells and in BAL were significantly higher (P < 0.01) in sub-unit compared with killed whole cell vaccinees. IgA was not detected in BAL from intra-muscularly dosed sub-unit vaccinees and thus the protection achieved against inhalational challenge with Yersinia pestis is attributed to the induction of systemic immunity to both the F1 and V antigens in the sub-unit vaccine. The enhanced protective efficacy of this sub-unit vaccine over an existing vaccine has been demonstrated in an animal model of pneumonic plague.

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.

The Yersinia pestis pH 6 antigen was expressed by, and purified from, Escherichia coli containing cloned psa genes. By an enzyme-linked immunosorbence-based assay, purified pH 6 antigen bound to gangliotetraosylceramide (GM1A), gangliotriaosylceramide (GM2A), and lactosylceramide (LC) (designations follow the nomenclature of L. Svennerholm [J. Neurochem. 10:613-623, 1963]). Binding to GM1A, GM2A, and LC was saturable, with 50% maximal binding occurring at 498 +/- 4, 390, and 196 +/- 3 nM, respectively. Thin-layer chromatography (TLC) overlay binding confirmed that purified pH 6 antigen bound to GM1A, GM2A, and LC and also revealed binding to hydroxylated galactosylceramide. Intact E. coli cells which expressed the pH 6 antigen had a specificity similar to that of purified pH 6 in the TLC overlay assay except that nonhydroxylated galactosylceramide was also bound. The binding patterns observed indicate that the presence of beta1-linked galactosyl residues in glycosphingolipids is the minimum determinant required for binding of the pH 6 antigen.

The efficacy of doxycycline and ciprofloxacin against an experimental tularaemia infection was assessed by comparing the median lethal dose (MLD) of Francisella tularensis Schu4 biotype a strain given intraperitoneally to antibiotic-treated and untreated mice. In untreated Porton outbred mice this was

(1998). The detection of insertion sequences within the human pathogen Burkholderia pseudomallei which have been identified previously in Burkholderia cepacia. FEMS Microbiol Lett, 162(1), 69-74.

Using primers designed from the nucleotide sequences of five insertion elements identified previously in Burkholderia cepacia, the presence of two insertion sequences (IS406 and IS407) was detected in chromosomal DNA isolated from strains of the human pathogen Burkholderia pseudomallei. The IS407 homologue was cloned from B. pseudomallei NCTC 4845 and nucleotide sequenced to confirm its identity and degree of homology with B. cepacia IS407. A PCR amplification product from B. pseudomallei NCTC 4845 DNA provided an IS407 probe which was used to determine, by Southern blotting, the number and location of copies of IS407 in ten strains of B. pseudomallei and four representatives from three of the five genomovars of B. cepacia.

Abstract: The detection of insertion sequences within the human pathogen Burkholderia pseudomallei which have been identified previously in Burkholderia cepacia.

Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

(1998). Structure and function of clostridial A-toxin and its role in gas-gangrene. ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 216, U66-U66. Author URL

A panel of ten site-directed mutants of Clostridium perfringens epsilon-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native epsilon-toxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated epsilon-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant epsilon-toxin.

Yersinia pestis, the causative organism of plague, produces a capsular protein (fraction 1 or F1 antigen) that is one of the major virulence factors of the bacterium. We report here the production, structural and immunological characterisation of a recombinant F1 antigen (rF1). The rF1 was purified by ammonium sulfate fractionation followed by FPLC Superose gel filtration chromatography. Using FPLC gel filtration chromatography and capillary electrophoresis, we have demonstrated that rF1 antigen exists as a multimer of high molecular mass. This multimer dissociates after heating in the presence of SDS and reassociation occurs upon the removal of SDS. Using circular dichroism, we have monitored the reassociation of monomeric rF1 into a multimeric form. Mice immunised with monomeric or multimeric rF1 develop similar immune responses, but mice immunised with monomeric rF1 were significantly less well protected against a challenge of 1 x 10(6) cfu of Y. pestis than mice immunised with multimeric rF1 (1/7 compared with 5/7). The significance of this result in terms of the structure and the function of rF1 is discussed.

We have developed a new two-step method for targeting cytotoxic drugs to tumour cells. The method firstly involves the binding to tumour cells of antibody-phospholipase C immunoconjugates or fusion proteins. Further to washing or clearance of the immunoconjugates, liposomes are introduced which are specifically lysed at the tumour site by PLC to release their cytotoxic contents in the vicinity of the tumour cells. For two alternative human cell lines, a synergistic inhibition of cell proliferation was seen for combined treatment with a specific immunoconjugate and daunorubicin encapsulated liposomes. For tumour xenografts in mice, the combined treatment resulted in an inhibition of tumour growth although with no eradication of tumours at the doses used. The two-step antibody-PLC/liposome approach offers broad possibilities for the precise delivery of payloads of cytotoxic drugs to tumour sites.

The efficacies of ciprofloxacin and doxycycline prophylaxis and therapy were assessed against experimental pneumonic plague infections induced by two strains of Yersinia pestis in a mouse model. When exposed to an aerosol of Y. pestis strain GB, containing 8.39 x 10(5) +/- 4.17 x 10(4) cfu, the retained dose was 7.3 x 10(3) +/- 2.3 x 10(3) cfu. When exposed to an aerosol of Y. pestis strain CO-92, containing 1.86 x 10(5) +/- 7.4 x 10(3) cfu, the retained dose was 3.4 x 10(4) +/- 2.6 x 10(3) cfu. Both strains resulted in a respiratory and systemic infection closely resembling human pneumonic plague. Ciprofloxacin prophylaxis and therapy was successful against both strains for up to 24 h after challenge, but not after 48 h. Both doxycycline prophylaxis and therapy regimens were ineffective against both strains, although strain CO-92 was more susceptible in vitro to doxycycline than strain GB and supra-MIC levels were achieved in the serum and lungs of the animal.

The alpha-toxin of Clostridium perfringens is the major virulence determinant for gas gangrene in man. The gene encoding the alpha-toxin has been cloned into E. coli from two strains of the bacterium (NCTC8237 and CER89L43) and subsequently purified to homogeneity. The two strains of alpha-toxin differ by five amino acids, resulting in the toxin from NCTC8237 being sensitive to chymotrypsin digestion while that from CER89L43 is resistant. The alpha-toxin from each of these strains has been crystallized in two different forms by the hanging-drop vapour-diffusion method at 293 K. CER89L43 form I crystals belong to space group R32 and have two molecules in the crystallographic asymmetric unit and a unit cell with a = b = 151.4, c = 195.5 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffracted to dmin = 1.90 A. The characteristics of the NCTC8237 form I crystals have already been reported. The form II crystals from both strains belong to space group C2221 with one molecule in the crystallographic asymmetric unit and, for strain CER89L43, have cell dimensions a = 61.05, b = 177.50, c = 79.05 A, alpha = beta = gamma = 90 degrees, while for strain NCTC8237 the cell dimensions are a = 60.50, b = 175.70, c = 80.20 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to maximum resolutions of 1.85 and 2.1 a for the CER89L43 and the NCTC8237 strains, respectively.

The phospholipases are a diverse group of enzymes, produced by a variety of Gram-positive and Gram-negative bacteria. The roles of these enzymes in the pathogenesis of infectious disease is equally diverse. It is only recently that molecular genetic approaches have allowed data to be obtained which indicates the role of these enzymes in the disease process. In the case of some pathogens phospholipases play an overriding role in disease. Roles for these enzymes have been demonstrated in the pathogenesis of disease caused by extracellular and intracellular pathogens and by disease caused by pathogens which enter via the respiratory tract, the intestinal tract or after traumatic injury. Some of the mechanisms by which phospholipases C affect tissues in vitro or ex vivo are understood but, in the main, the mechanisms by which phospholipases C affect tissues in vivo are not known. A key event, which can determine the extent of involvement of phospholipases in the disease process, is the interaction of the enzyme with phospholipids in eukaryotic cell membranes. Whilst progress has been made in understanding the molecular basis of these interactions, the process is far from understood. Two theories attempt to explain the reasons why only some phospholipases C are membrane active. In general, the membrane active enzymes are able to hydrolyse both phosphatidylcholine and sphingomyelin and appear to have mechanisms which allow them to interact with membrane phospholipids. The structural differences between phosphatidylcholine and sphingomyelin lie within the fatty acyl chain/ester bond region which would be partially embedded in the membrane bilayer. Therefore, there may be a common explanation for membrane interaction and recognition of both phospholipid types. The value of this information will be several fold. The demonstration of the role of these enzymes in disease will allow the development of vaccines or therapeutics which block the effects of these enzymes. In this context it is worth bearing in mind that eukaryotic phospholipases C, which play key roles in many inflammatory and autoimmune diseases, are the subject of intense study by the pharmaceutical industry. Some of the bacterial toxins are potent cytotoxic agents and this has encouraged some workers to explore the possibility that immunotoxins can be developed (Chovnick et al. 1991). Purified recombinant phospholipases C will continue to be used in the study of cell membranes, and the increasing numbers of enzymes with different substrate specificities will enhance their application.

The phospholipases are a diverse group of enzymes, produced by a variety of Gram-positive and Gram-negative bacteria. The roles of these enzymes in the pathogenesis of infectious disease is equally diverse. It is only recently that molecular genetic approaches have allowed data to be obtained which indicates the role of these enzymes in the disease process. In the case of some pathogens phospholipases play an overriding role in disease. Roles for these enzymes have been demonstrated in the pathogenesis of disease caused by extracellular and intracellular pathogens and by disease caused by pathogens which enter via the respiratory tract, the intestinal tract or after traumatic injury. Some of the mechanisms by which phospholipases C affect tissues in vitro or ex vivo are understood but, in the main, the mechanisms by which phospholipases C affect tissues in vivo are not known. A key event, which can determine the extent of involvement of phospholipases in the disease process, is the interaction of the enzyme with phospholipids in eukaryotic cell membranes. Whilst progress has been made in understanding the molecular basis of these interactions, the process is far from understood. Two theories attempt to explain the reasons why only some phospholipases C are membrane active. In general, the membrane active enzymes are able to hydrolyse both phosphatidylcholine and sphingomyelin and appear to have mechanisms which allow them to interact with membrane phospholipids. The structural differences between phosphatidylcholine and sphingomyelin lie within the fatty acyl chain/ester bond region which would be partially embedded in the membrane bilayer. Therefore, there may be a common explanation for membrane interaction and recognition of both phospholipid types. The value of this information will be several fold. The demonstration of the role of these enzymes in disease will allow the development of vaccines or therapeutics which block the effects of these enzymes. In this context it is worth bearing in mind that eukaryotic phospholipases C, which play key roles in many inflammatory and autoimmune diseases, are the subject of intense study by the pharmaceutical industry. Some of the bacterial toxins are potent cytotoxic agents and this has encouraged some workers to explore the possibility that immunotoxins can be del eloped (Chovnick et al. 1991). Purified recombinant phospholipases C will continue to be used in the study of cell membranes, and the increasing numbers of enzymes with different substrate specificities will enhance their application.

The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective.

Type III-mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic Yersinia. LcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti-LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV-induced protective immunity.

In this study, we have shown that severe combined immunodeficient/beige mice reconstituted with hyperimmune Balb/c lymphocytes can be used as a model to demonstrate adoptive and passive protection against plague infection. Reconstitution of severe combined immunodeficient/beige mice was successful in nine out of ten mice as demonstrated by spleen colonisation and sustained circulating immunoglobulin titres. Furthermore, an increase in antibody titre was evident after a booster immunisation of reconstituted mice. Presence of circulating antibody correlated with protection against a systemic plague challenge and indicated that in reconstituted mice adoptive transfer of a functional immune system had occurred. The severe combined immunodeficient/beige mouse was also used to demonstrate passive protection against inhaled and systemic plague infection. The reconstituted severe combined immunodeficient/beige mouse model demonstrating protective immunity against plague will be further developed to identify the immune cell subsets responsible for this protection.

The gene encoding the alpha-(cpa) is present in all strains of Clostridium perfringens, and the purified alpha-toxin has been shown to be a zinc-containing phospholipase C enzyme, which is preferentially active towards phosphatidylcholine and sphingomyelin. The alpha-toxin is haemolytic as a result if its ability to hydrolyse cell membrane phospholipids and this activity distinguishes it from many other related zinc-metallophospholipases C. Recent studies have shown that the alpha-toxin is the major virulence determinant in cases of gas gangrene, and the toxin might play a role in several other diseases of animals and man as diverse as necrotic enteritis in chickens and Crohn's disease in man. In gas gangrene the toxin appears to have three major roles in the pathogenesis of disease. First, it is able to cause mistrafficking of neutrophils, such that they do not enter infected tissues. Second, the toxin is able to cause vasoconstriction and platelet aggregation which might reduce the blood supply to infected tissues. Finally, the toxin is able to detrimentally modulate host cell metabolism by activating the arachidonic acid cascade and protein kinase C. The molecular structure of the alpha-toxin reveals a two domain protein. The amino-terminal domain contains the phospholipase C active site which contains zinc ions. The carboxyterminal domain is a paralogue of lipid binding domains found in eukaryotes and appears to bind phospholipids in a calcium-dependent manner. Immunisation with the non-toxic carboxyterminal domain induces protection against the alpha-toxin and gas gangrene and this polypeptide might be exploited as a vaccine. Other workers have exploited the entire toxin as the basis of an anti-tumour system.

Recombinant vaccinia viruses that expressed the nontoxic C-domain of Clostridium perfringens alpha-toxin were constructed. The J2R (thymidine kinase [TK] gene) and B13R (serpin 2 [SPI-2] gene) loci were used as insertion sites for the clostridial DNA, and expression of the foreign protein was measured in each case. A double recombinant that encoded the alpha-toxin truncate at the B13R locus and the protective antigen of Bacillus anthracis at the J2R locus was also constructed. Although differences in expression of the alpha-toxin C-domain were recorded, all of the vaccinia recombinants protected mice against a lethal challenge with alpha-toxin demonstrating that a recombinant vaccinia virus can be used to provide protection against a toxin challenge that is known to be solely antibody mediated.

The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.

(1999). Differences in the carboxy-terminal (Putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes. Infect Immun, 67(7), 3297-3301.

The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.

Abstract: Differences in the carboxy-terminal (Putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes.

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd approximately 175-250 microM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods.

(1999). An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin Exp Immunol, 116(1), 107-114.

The objective of this study was to identify an immunological correlate of protection for a two-component subunit vaccine for plague, using a mouse model. The components of the vaccine are the F1 and V antigens of the plague-causing organism, Yersinia pestis, which are coadsorbed to alhydrogel and administered intramuscularly. The optimum molar ratio of the subunits was determined by keeping the dose-level of either subunit constant whilst varying the other and observing the effect on specific antibody titre. A two-fold molar excess of F1 to V, achieved by immunizing with 10 micrograms of each antigen, resulted in optimum antibody titres. The dose of vaccine required to protect against an upper and lower subcutaneous challenge with Y. pestis was determined by administering doses in the range 10 micrograms F1 + 10 micrograms V to 0.01 microgram F1 + 0.01 microgram V in a two-dose regimen. For animals immunized at the 1-microgram dose level or higher with F1 + V, an increase in specific IgG1 titre was observed over the 8 months post-boost and they were fully protected against a subcutaneous challenge with 10(5) colony-forming units (CFU) virulent Y. pestis at this time point. However, immunization with 5 micrograms or more of each subunit was required to achieve protection against challenge with 10(7) CFU Y. pestis. A new finding of this study is that the combined titre of the IgG1 subclass, developed to F1 plus V, correlated significantly (P < 0.05) with protection. The titres of IgG1 in vaccinated mice which correlated with 90%, 50% and 10% protection have been determined and provide a useful model to predict vaccine efficacy in man.

Abstract: An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model.

The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37 degrees C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.

A range of clostridial species produce phospholipases C. The zinc metallo phospholipases C have related sequences but different properties. All of these enzymes may be arranged, like alpha-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.

The growth of an aroA mutant of Salmonella typhimurium (SL3261) in minimal medium containing 0.5 M NaCl resulted in the intracellular accumulation of 2.2 micromol trehalose/mg total protein. The vacuum drying of these bacteria in the presence of trehalose allowed the recovery of 35% of the viable cells that were present before drying. In contrast, bacteria cultured in control medium accumulated 0.4 micromol trehalose/mg total protein and only 5% of the viable cells were recovered after vacuum drying with trehalose. Similar results were obtained when S. typhimurium SL3261, expressing the vaccine antigen (F1-antigen) of Yersinia pestis, was cultured in minimal medium with or without 0.5 M NaCl and dried in the presence of trehalose. Although these results indicate the potential for trehalose stabilisation of vaccine strains of S. typhimiurium, growth in minimal medium containing 0.5 M NaCl resulted in the loss of invasion competence of the bacteria.

Francisella tularensis is the etiological agent of tularemia, a serious disease in several Northern hemisphere countries. The organism has fastidious growth requirements and is very poorly understood at the genetic and molecular levels. Given the lack of data on this organism, we undertook the sample sequencing of its genome. A random library of DNA fragments from a highly virulent strain (Schu 4) of F. tularensis was constructed and the nucleotide sequences of 13,904 cloned fragments were determined and assembled into 353 contigs. A total of 1.83 Mb of nucleotide sequence was obtained that had a G+C content of 33.2%. Genes located on plasmids pOM1 and pNFL10, which had been previously isolated from low virulence strains of F. tularensis, were absent but all of the other known F. tularensis genes were represented in the assembled data. F. tularensis Schu4 was able to grow in the absence of aromatic amino acids and orthologues of genes which could encode enzymes in the shikimate pathway in other bacteria were identified in the assembled data. Genes that could encode all of the enzymes in the purine biosynthetic and most of the en- zymes in the purine salvage pathways were also identified. This data will be used to develop defined rationally attenuated mutants of F. tularensis, which could be used as replacements for the existing genetically undefined live vaccine strain.

Clostridium perfringens isolates are currently classified into one of five biotypes on the basis of the differential production of alpha-, beta-, epsilon- and iota-toxins. Different biotypes are associated with different diseases of man and animals. In this study a multiple PCR assay was developed to detect the genes encoding these toxins. In addition, detection of the genes encoding the C. perfringens enterotoxin and beta2-toxin allowed subtyping of the bacteria. C. perfringens isolates taken from a variety of animals, including foals, piglets or lambs, were genotyped using this assay. Most of the isolates were found to be genotype a and the gene encoding beta2-toxin [corrected] was present in 50% of the isolates genotyped. A significant association between C. perfringens possessing the beta2-toxin gene and diarrhoea in piglets was identified, suggesting that beta2-toxin may play a key role in the pathogenesis of the disease.

The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens. For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines. A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs. An isogenic Y. pestis htrA mutant was constructed using a reverse genetics approach. In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model. However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.

Ferrets which had been orally dosed with 5 mg of Staphylococcal enterotoxin B (SEB) responded with an increase in subcutaneous temperature. At 75 min, the subcutaneous temperature was significantly higher (+ 0.9 degrees C +/- 0.38 degrees C, P < 0.007) than in control animals. Animals dosed with 1 or 2 mg of SEB responded with a small, but not significant, increase in subcutaneous temperature. All of the animals dosed with 5 mg of SEB retched and vomited. The mean latency for the onset of retching was 105 +/- 36 min, and the mean latency for the onset of vomiting was 106 +/- 34 min. The mean number of retches was 17.8 +/- 19.6, and the mean number of vomits was 2.0 +/- 1.5. These findings indicate that ferrets can be used as alternatives to primates for the study of the biological activities of SEB.

(2000). Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities. Arch Biochem Biophys, 384(1), 24-30.

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

Abstract: Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.

The soil taken from various areas of a former chemical weapons factory, located at Locknitz, Mecklenburg-Vorpommem in Germany was shown to be contaminated with arsine oil and its breakdown products. The total number of cells and the number of culturable bacterial cells were similar in soil samples taken from arsine free or arsine-contaminated areas of the site and were similar at different depths, up to 55 cm, from the soil surface. However, culturable arsenic-resistant bacteria were found in greater numbers in arsine-contaminated soil (mean 1.56x105 cfu/g) than in uncontaminated soil (mean 4x104 cfu/g). Seventy-four per cent of the arsenic resistant bacteria were Gram-negative and the predominant species were shown to be Pseudomonas corrugata and Pseudomonas tolaasii.

A recombinant vaccine candidate was developed that protected mice against botulinum neurotoxin serotype F (BoNTF) intoxication. A synthetic gene encoding BoNTF fragment C (rBoNTF(H(c))) was designed, constructed, and inserted into a plasmid for expression in the yeast Pichia pastoris. A total cell protein content of 2.9 g was obtained per liter of fermentation broth. Recombinant rBoNTF(H(c)) was purified from the soluble yeast extract in two chromatographic steps. The process employed Mono S cation exchange chemistry followed by Alkyl-Superose hydrophobic interaction chromatography, producing material judged to be greater than 98% pure by SDS-PAGE. The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste. rBoNTF(H(c)) was also purified from the insoluble fraction of the yeast cell lysate. Because the fragment C in the pellet was 35% of the total insoluble protein, only a Mono S cation exchange chromatography step was necessary to achieve a purity greater than 98%. Mice that received three injections of 0.2 microgram of purified soluble rBoNTF(H(c)) were completely protected when challenged with 1000 mouse ip LD(50) of BoNTF toxin. Similarly, three doses of 1 microgram of purified resolubilized rBoNTF(H(c)) completely protected mice from a challenge of 5000 mouse ip LD(50) of BoNTF toxin. Individual serum antibody ELISA titers of mice injected with soluble rBoNTF(H(c)) correlated with survival as all 34 mice with ELISA titers of 100 or greater survived toxin challenge. The work presented here demonstrates that purified rBoNTF(H(c)) is able to protect against a high challenge dose of neurotoxin.

A synthetic gene encoding the Hc (binding) domain of Clostridium botulinum neurotoxin F (FHc) was expressed in Escherichia coli fused to maltose binding protein (MBP). The purified MBP-FHc and FHc isolated after removal of MBP were evaluated in mice for their ability to protect against toxin challenge. Balb/c mice developed a protective immune response following administration of either protein via the intraperitoneal or intramuscular routes. A comparison of antibody titres and protection following single and multiple vaccinations and the effects of dosage are shown. The long term protection afforded by the vaccines was also investigated. Ten months following vaccination mice were still protected when challenged with 10(4) MLD(50) doses of botulinum toxin F.

Attenuated mutants of Salmonella typhimurium are being evaluated as delivery systems for a variety of heterologous vaccine antigens. Gene promoters which are induced in vivo can direct the stable expression of genes encoding these antigens. We have investigated the utility of the phoP, ompC, pagC and lacZ gene promoters for expression of the Y. pestis F1-antigen in S. typhimurium SL3261 (aroA). After i.g. (intragastric) dosing the highest level of spleen colonisation was found with recombinant Salmonella expressing F1-antigen from the phoP gene promoter, and this recombinant was most effective in inducing serum and mucosal antibody responses. The use of the pagC gene promoter to direct expression of F1-antigen resulted in the induction of serum and mucosal antibody responses even though the recombinant Salmonella were unable to colonise spleen tissues suggesting that colonisation of these tissues is not essential for the induction of antibody responses.

The epithelial Madin Darby canine kidney (MDCK) cell line and 17 human cell lines were examined for sensitivity to Clostridium perfringens type D epsilon-toxin. MDCK cells were confirmed as being sensitive to the toxin. In addition, the Caucasian renal leiomyoblastoma (G-402) human cell line was identified as being epsilon-toxin sensitive. Using the MTS/PMS assay system the concentration of toxin reducing cell culture viability by 50% (LC50) was found to be 2 microg/ml in MDCK cells. The LC50 for G-402 cells was 280 microg/ml. Epsilon-Toxin was found to be rapid acting in MDCK cells exposed to a maximum lethal dose of the toxin (40% loss of viability after a 0.5 h exposure), but slower acting in G-402 cells (40% loss of viability after 1.7 h exposure). Photomicrography of toxin exposed cultures indicated necrotic cell death on exposure to epsilon-toxin. Investigations using an antibody probe indicated that epsilon-toxin could bind to many cell surface proteins in both MDCK, G-402 and a toxin insensitive human cell line (CAKI-2). It has previously been found that the toxin may bind to the cell surface via glycosylated moieties. However, exposing MDCK and G-402 cells to epsilon-toxin in the presence of sialic acid and several different sugars did not reduce the lethal effects of the toxin.

In this study, the protection afforded against aerosolised Yersinia pestis by injection of a single dose of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine, prepared by admixing F1 antigen derived from a Y. pestis cell culture supernatant with recombinant V antigen derived from an E. coli cell lysate, fully protected an outbred strain of mouse against exposure to 10(6) CFU of virulent plague organisms (10(4) mouse lethal doses, MLD). In contrast, the whole cell vaccine provided only 16% protection against the same level of challenge. Furthermore, sub-unit vaccinees were able to clear the bacteria from their lungs post-challenge whereas bacteria were cultured from the lungs of a surviving KWC vaccinee post-challenge. In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable and the levels of F1-specific IgG in serum and in broncho-alveolar washings were significantly lower (p

Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.

Yersinia pestis is the etiological agent of bubonic and pneumonic plague, diseases which have caused over 200 milllion human deaths in the past. Plague still occurs throughout the world today, though for reasons that are not fully understood pandemics of disease do not develop from these outbreaks. Antibiotic treatment of bubonic plague is usually effective, but pneumonic plague is difficult to treat and even with antibiotic therapy death often results. A killed whole cell plague vaccine has been used in the past, but recent studies in animals have shown that this vaccine offers poor protection against pneumonic disease. A live attenuated vaccine is also available. Whilst this vaccine is effective, it retains some virulence and in most countries it is not considered to be suitable for use in humans. We review here work to develop improved sub-unit and live attenuated vaccines against plague. A sub-unit vaccine based on the F1- and V-antigens is highly effective against both bubonic and pneumonic plague, when tested in animal models of disease. This vaccine has been used to explore the utility of different intranasal and oral delivery systems, based on the microencapsulation or Salmonella delivery of sub-units.

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.

(2001). The failure of different strains of Yersinia pestis to produce lipopolysaccharide O-antigen under different growth conditions is due to mutations in the O-antigen gene cluster. FEMS Microbiol Lett, 197(2), 229-233.

The lipopolysaccharide (LPS) from eight strains of Yersinia pestis which had been cultured at 28 degrees C appeared to be devoid of an O-antigen when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LPS isolated from three of these strains which had been cultured at 37 degrees C also appeared to be devoid of an O-antigen. When the LPS from Y. pestis strain CO92 was purified and analysed by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry, the observed signals were in the mass range predicted for molecules containing lipid a plus the core oligosaccharide but lacking an O-antigen. The nucleotide sequence of Y. pestis strain CO92 revealed the presence of a putative O-antigen gene cluster. However, frame-shift mutations in the ddhB, gmd, fcl and ushA genes are likely to prevent expression of the O-antigen thus explaining the loss of phenotype.

Abstract: The failure of different strains of Yersinia pestis to produce lipopolysaccharide O-antigen under different growth conditions is due to mutations in the O-antigen gene cluster.

Mice immunised with lipopolysaccharide (LPS) from Francisella tularensis were protected against challenge with the live vaccine strain (LVS). However, when similarly immunised mice were challenged using the fully virulent F. tularensis strain Schu4, only an increase in the time to death was observed. Passive transfer of serum from LPS-immunised mice to naive mice afforded protection against F. tularensis LVS. LPS-immunised mice depleted of either CD4+ or CD8+ T-cells survived a F. tularensis LVS challenge although the rate of clearance of bacteria from the spleen was significantly reduced in the CD8+ depleted group. LPS-immunised mice boosted with F. tularensis LVS were re-challenged with F. tularensis Schu4. This cohort was significantly protected (LD(50) increased from 1000 CFU). However, passive transfer of serum did not confer protection and mice depleted of CD4+ or CD8+ T-cells did not survive.

Using a combination of nanoflow-electrospray ionization and time-of-flight mass spectrometry we have analyzed the oligomeric state of the recombinant V antigen from Yersinia pestis, the causative agent of plague. The mass spectrometry results show that at pH 6.8 the V antigen in solution exists predominantly as a dimer and a weakly associated tetramer. A monoclonal antibody 7.3, raised against the V antigen, gave rise to mass spectra containing a series of well-resolved charge states at m/z 6000. After addition of aliquots of solution containing V antigen in substoichiometric and molar equivalents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the antibody-antigen complex results in the selective release of the dimer from the complex supporting the proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the antibody is specific for the V antigen because no complex with F1 was detected even in the presence of a 10-fold molar excess of F1 antigen. More generally this work demonstrates a rapid means of assessing antigen subunit interactions as well as the stoichiometry and specificity of binding in antibody-antigen complexes.

Mass spectrometry has been used to examine the subunit interactions in the capsular F1 antigen from Yersinia pestis, the causative agent of the plague. Introducing the sample using nanoflow electrospray from solution conditions in which the protein remains in its native state and applying collisional cooling to minimize the internal energy of the ions, multiple subunit interactions have been maintained. This methodology revealed assemblies of the F1 antigen that correspond in mass to both 7-mers and 14-mers, consistent with interaction of two seven-membered units. The difference between the calculated masses and those measured experimentally for these higher-order oligomers was found to increase proportionately with the size of the complex. This is consistent with a solvent-filled central cavity maintained on association of the 7-mer to the 14-mer. The charge states of the ions show that an average of one and four surface accessible basic side-chains are involved in maintaining the interactions between the 7-mer units and neighboring subunits, respectively. Taken together, these findings provide new information about the stoichiometry and packing of the subunits involved in the assembly of the capsular antigen structure. More generally, the data show that the symmetry and packing of macromolecular complexes can be determined solely from mass spectrometry, without any prior knowledge of higher order structure

Lipopolysaccharide (LPS) extracted from eight strains of Yersinia pestis, which had been cultured at 28 or 37 degrees C, reacted equally well, in Western blots, with four monoclonal antibodies generated against the LPS from a single strain of Y. pestis cultured at 28 degrees C. LPS was extracted and purified from Y. pestis strain GB, which had been cultured at 28 degrees C. When the LPS was analysed by SDS-PAGE and MALDI-TOF mass spectrometry it was found to be devoid of an O-antigen. The LPS possessed activity of 2.7 endotoxin units/ng in the Limulus amoebocyte lysate assay. The LPS stimulated the production of TNFalpha and IL-6 from mouse macrophages, but was less active in these assays than LPS isolated from Escherichia coli strain 0111. Y. pestis LPS, either alone or with cholera toxin B subunit, was used to immunize mice. Either immunization schedule resulted in the development of an antibody response to LPS. However, this response did not provide protection against 100 MLD of Y. pestis strain GB.

Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase a (PldA) mutant exh