Search

Melioidosis is a severe infectious disease of animals and humans caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei. An Inv/Mxi-Spa-like type III protein secretion apparatus, encoded by the B. pseudomallei bsa locus, facilitates bacterial invasion of epithelial cells, escape from endocytic vesicles and intracellular survival. This study investigated the role of the Bsa type III secretion system in the pathogenesis of melioidosis in murine models. B. pseudomallei bipD mutants, lacking a component of the translocation apparatus, were found to be significantly attenuated following intraperitoneal or intranasal challenge of BALB/c mice. Furthermore, a bipD mutant was attenuated in C57BL/6 IL-12 p40−/− mice, which are highly susceptible to B. pseudomallei infection. Mutation of bipD impaired bacterial replication in the liver and spleen of BALB/c mice in the early stages of infection. B. pseudomallei mutants lacking either the type III secreted guanine nucleotide exchange factor BopE or the putative effectors BopA or BopB exhibited varying degrees of attenuation, with mutations in bopA and bopB causing a significant delay in median time to death. This indicates that bsa-encoded type III secreted proteins may act in concert to determine the outcome of B. pseudomallei infection in mice. Mice inoculated with the B. pseudomallei bipD mutant were partially protected against subsequent challenge with wild-type B. pseudomallei. However, immunization of mice with purified BipD protein was not protective.

Heat shock proteins (Hsps) have attracted significant attention as protective antigens against a range of diseases caused by bacterial pathogens. However, more recently there have been suggestions that the protective response is due to the presence of peptide components other than Hsps. We have shown that mice that had been immunized with purified heat shock protein 60 (Hsp60) isolated fromFrancisella tularensiswere protected against a subsequent challenge with some strains of the bacterium. However, this protection appeared to be due to trace amounts of lipopolysaccharide, which were too low to be detected by using theLimulusamoebocyte lysate assay. This finding raises the possibility that the protection afforded by other bacterial Hsp60 proteins may be due to trace quantities of polysaccharide antigens carried by and acting in conjunction with the Hsps.

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.

Yersinia pestis is the aetiological agent of plague, a disease that has a place in history as one the major causes of death from the 14th to the 17th Centuries1. It is estimated that, during the Black Death pandemic, approximately 30% of the population of Europe died of plague, and so great in number were the corpses that, in many parts of Europe, the dead were placed in burial pits rather than receiving individual burials. Y. pestis has also been responsible for two other pandemics of disease. The first of these, the Justinian plague, occurred during the 1st Century. The third pandemic occurred during the latter part of the 19th Century and was confined mainly to South-East Asia1. Even today, several thousand cases of plague are reported to the World Health Organization each year, mainly from South-East Asia, the southwestern parts of the USA, Madagascar and Africa.

Clostridium perfringens phospholipase C (Cp-PLC), the major virulence factor in the pathogenesis of gas gangrene, is a Zn(2+) metalloenzyme with lecithinase and sphingomyelinase activities. Its structure shows an N-terminal domain containing the active site, and a C-terminal Ca(2+) binding domain required for membrane interaction. Although the knowledge of the structure of Cp-PLC and its interaction with aggregated phospholipids has advanced significantly, an understanding of the effects of Cp-PLC in mammalian cells is still incomplete. Cp-PLC binds to artificial bilayers containing cholesterol and sphingomyelin or phosphatidylcholine (PC) and degrades them, but glycoconjugates present in biological membranes influence its binding or positioning toward its substrates. Studies with Cp-PLC variants harboring single amino-acid substitutions have revealed that the active site, the Ca(2+) binding region, and the membrane interacting surface are required for cytotoxic and haemolytic activity. Cp-PLC causes plasma membrane disruption at high concentrations, whereas at low concentrations it perturbs phospholipid metabolism, induces DAG generation, PKC activation, Ca(2+) mobilization, and activates arachidonic acid metabolism. The cellular susceptibility to Cp-PLC depends on the composition of the plasma membrane and the capacity to up-regulate PC synthesis. The composition of the plasma membrane determines whether Cp-PLC can bind and acquire its active conformation, and thus the extent of phospholipid degradation. The capacity of PC synthesis and the availability of precursors determine whether the cell can replace the degraded phospholipids. Whether the perturbations of signal transduction processes caused by Cp-PLC play a role in cytotoxicity is not clear. However, these perturbations in endothelial cells, platelets and neutrophils lead to the uncontrolled production of intercellular mediators and adhesion molecules, which inhibits bacterial clearance and induces thrombotic events, thus favouring bacterial growth and spread in the host tissues.

To investigateYersiniapathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organismCaenorhabditis eleganswas studied. Three strains ofYersinia pestis, including a strain lacking pMT1, caused blockage and death ofC. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains ofYersinia enterocoliticacaused no effect. Strains ofYersinia pseudotuberculosisshowed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9·5 % an intermediate infection, and 9·5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection ofC. elegansand those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants ofY. pseudotuberculosisstrain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 definedC. elegansmutants identified four where mutation caused (complete) resistance to infection byY. pseudotuberculosisYPIII pIB1. These mutants,srf-2,srf-3,srf-5and the dauer pathway genedaf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.

The epithelial Madin Darby canine kidney (MDCK) cell line, Caucasian renal leiomyoblastoma (G-402) cells, human small airways epithelial (HSAE) cells, human bronchial epithelial (HBE) cells and human renal proximal tubule (HRPT) epithelial cells were examined for sensitivity to Clostridium perfringens biotype D o-toxin. MDCK and G-402 cells were confirmed as being the only established cell lines that are sensitive to the toxin. HSAE, HBE and HRPT epithelial cells were only found to be sensitive to the toxin at concentrations of -1 mg/ mL. Cultures of MDCK and G-402 cells, with increased resistance (tolerance) to the cytotoxic effects of o-toxin, were developed by exposing these cultures to progressively higher concentrations of toxin. The greatest relative increase in tolerance to o-toxin was developed in MDCK cells, in which the LC50 in control cultures was 2mg/mL as determined by the MTS/PMS assay system; after selection for tolerance, this was raised to 100 mg/mL. This represents a 50-fold increase in tolerance as measured by this index. Using G-402 cells, it was possible to increase the LC50 by twofold from 290 to 590 mg/mL. Subsequent 2-D electrophoresis of membrane preparations from tolerant and control MDCK cells revealed that the expression of a discrete group of proteins found in control cells with a range of molecular weights from 32–36 kDa, all with acidic isoelectric points (IEPs), were either not expressed in o-toxin tolerant cells or had undergone a shift in IEP to a more alkaline pH in tolerant cells. This suggests that o-toxin lethality in MDCK cells may be mediated by membrane-located proteins. Their absence or alteration in toxin-resistant cells would, at least partly, explain the failure of most cell lines to demonstrate sensitivity to this toxin, despite being derived from tissues that are damaged by o-toxin. This approach may have utility in the study of other toxin / cell interactions and could be used in the development of novel medical countermeasures by identifying cellular targets which mediate toxin lethality.

Francisella tularensis is an intracellular pathogen with a very low infectious dose for humans. Several forms of tularaemia occur, which range from a severely debilitating to a fatal disease. Diagnosis is difficult due to the generalised, nonspecific nature of symptoms and the difficulty in culturing the slow-growing and nutritionally fastidious pathogen. A live attenuated vaccine strain (LVS) has been used in humans as an investigational new drug and does appear to induce a protective response. However, the licensing of this vaccine has not yet been possible. For this reason, modern molecular biology approaches are being used in an attempt to devise replacement vaccines which may be more easily licensed. The approaches which are currently being considered include the production of subunit vaccines and the development of defined isogenic attenuated mutant strains of F. tularensis.

Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine.

All strains ofYersinia pestisexamined have been found to lack an O-antigen. In other members of theEnterobacteriaceae, the rough phenotype often results in attenuation. However,Y. pestisis the aetiological agent of bubonic plague. In evolving from the ancestral enteropathogenicYersinia pseudotuberculosis, and with the development of an arthropod-vectored systemic pathogenesis, smooth LPS production is not necessary forY. pestisvirulence and the metabolic burden has been alleviated by inactivation of the O-antigen biosynthetic operon. To investigate this,Y. pestisstrain KIM D27 was transformed with a plasmid carrying the operon encoding the O-antigen ofYersinia enterocoliticaO : 3. Expression of the O-antigen could be detected in silver-stained gels. The receptor for bacteriophage ϕYeO3-12 has been shown to be O-antigen, and infection by this bacteriophage results in lysis ofY. enterocoliticaO : 3. Expression of the O-antigen inY. pestisconferred sensitivity to lysis by ϕYeO3-12. The O-antigen-expressing clone was shown to be as virulent in mice by the intravenous route of challenge as the rough wild-type. Assays showed no alteration in the ability ofY. pestisto resist lysis by cationic antimicrobial peptides, serum or polymyxin.

Monoclonal antibodies specific for Yersinia pestis V antigen and F1 antigen, administered singly or in combination, protected mice in models of bubonic and pneumonic plague. Antibodies showed synergy when administered prophylactically and as a therapy 48 h postinfection. Monoclonal antibodies therefore have potential as a treatment for plague.

The utility of the htrA , pagC and nirB promoters to direct the expression of the carboxy-terminal (H C ) fragment of botulinum toxin F (FH C ) in Salmonella enterica var Typhimurium has been evaluated. Only low levels of serum antibody were induced after immunisation, and some protection against botulinum toxin type F was demonstrated after oral immunisation of mice with two doses of any of these recombinant Salmonella . Immunisation with two doses of recombinant Salmonella expressing FH C from the htrA promoter gave the greatest protection, against up to 10,000 mouse lethal doses of botulinum toxin type F. These results demonstrate the feasibility of an orally delivered vaccine against botulinum toxin type F.

Yersinia pestis is the aetiological agent of plague, a disease of humans that has potentially devastating consequences. Evidence indicates that Y. pestis evolved from Yersinia pseudotuberculosis, an enteric pathogen that normally causes a relatively mild disease. Although Y. pestis is considered to be an obligate pathogen, the lifestyle of this organism is surprisingly complex. The bacteria are normally transmitted to humans from a flea vector, and Y. pestis has a number of mechanisms which allow survival in the flea. Initially, the bacteria have an intracellular lifestyle in the mammalian host, surviving in macrophages. Later, the bacteria adopt an extracellular lifestyle. These different interactions with different host cell types are regulated by a number of systems, which are not well characterized. The availability of the genome sequence for this pathogen should now allow a systematic dissection of these regulatory systems.

Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location.

We describe the use of two insertion sequence elements (ISFtu1 and ISFtu2) in Francisella tularensis to type strains by restriction fragment length polymorphism (RFLP). The RFLP profiles of 17 epidemiologically unrelated isolates were determined and compared. Our results showed that RFLP profiles can be used to assign F. tularensis strains into five main groups corresponding to strains of F. tularensis subsp. tularensis , F. tularensis strain ATCC 6223, strains of F. tularensis subsp. holarctica , strains of F. tularensis subsp. holarctica from Japan, and F. tularensis subsp. mediaasiatica . The results confirm the genetic identities of these subspecies and also support the suggestion that strains of F. tularensis subsp. holarctica from Japan should be considered members of a separate biovar. These findings should support future studies to determine the genetic differences between strains of F. tularensis at the whole-genome level.

The type III secretion system is used by pathogenic Yersinia to translocate virulence factors into the host cell. A key component is the multifunctional LcrV protein, which is present on the bacterial surface prior to host cell contact and up-regulates translocation by blocking the repressive action of the LcrG protein on the cytosolic side of the secretion apparatus. The functions of LcrV are proposed to involve self-interactions (multimerization) and interactions with other proteins including LcrG. Coiled-coil motifs predicted to be present are thought to play a role in mediating these protein-protein interactions. We have purified recombinant LcrV, LcrG, and site-directed mutants of LcrV and demonstrated the structural integrity of these proteins using circular dichroism spectroscopy. We show that LcrV interacts both with itself and with LcrG and have obtained micromolar and nanomolar affinities for these interactions, respectively. The effects of LcrV mutations upon LcrG binding suggest that coiled-coil interactions indeed play a significant role in complex formation. In addition, comparisons of secretion patterns of effector proteins in Yersinia, arising from wild type and mutants of LcrV, support the proposed role of LcrG in titration of LcrV in vivo but also suggest that other factors may be involved.

Using a transposon mutagenesis approach, we have identified a mutant ofBurkholderia pseudomalleithat is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted theilvIgene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-typeB. pseudomallei.

Attenuated strains of Salmonella enterica serovar Typhimurium are used as carriers of heterologous antigens as candidate oral vaccines and, more recently, as carriers of DNA vaccines. In this study, recombinant Salmonella strains that were altered in their ability to colonise murine tissues in vivo when compared to parent strains were not, however, equally altered in their ability to invade murine cells in vitro. These results suggest that in vitro invasion studies may not be a representative model for colonisation of tissues in vivo, and that in vitro studies should ideally be used in conjunction with in vivo studies for the assessment of potential Salmonella vaccines.

A Burkholderia pseudomallei mutant which was attenuated in a mouse model of melioidosis was identified by a signature tagged mutagenesis approach. The transposon was shown to be inserted into a gene within the capsular biosynthetic operon. Compared with the wild-type bacteria this mutant demonstrated a 10(5)-fold increase in the median lethal dose in a mouse model and it did not react with a monoclonal antibody against high mol. wt polysaccharide of B. pseudomallei. To determine the kinetics of infection, mice were dosed intraperitoneally (i.p.) and intravenously (i.v.) with mutant and wild-type bacteria. After i.p challenge, the number of mutant bacteria in the peritoneal cavity declined, whereas wild-type bacteria proliferated. When administered by the i.v. route, the mutant was able to cause disease but the time to death was increased compared with the wild type. Mice were dosed with the mutant and subsequently challenged with wild-type B. pseudomallei, but the mutant failed to induce a protective immune response.

Dangerous pathogens are defined by the UK Health and Safety Executive's advisory committee as category 3 (those which cause severe human disease for which prophylaxis or therapy is usually available) or category 4 (as for category 3, but for which prophylaxis or therapy is not available). Research and development of vaccines for such pathogens is challenging, due to the safety constraints in the manipulation of these pathogens. This chapter discusses the various approaches which can be taken to develop candidate vaccines for these pathogens, including the potential impact of genome sequencing on shortening the time required for R&D. For these pathogens, a direct test of the efficacy of the candidate vaccines in man is not ethical and, therefore, particular emphasis is placed on the demonstration of efficacy in animal models. Emphasis is also placed on the derivation of surrogate markers of efficacy and a demonstration that these correlate with protection in the animal model.

The otsA and otsB genes, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase respectively, have been isolated from Salmonella enterica serovar typhimurium and nucleotide-sequenced. Induction of trehalose biosynthesis by exposure of bacteria to high osmotic strength resulted in the intracellular accumulation of trehalose. An otsA mutant of S. enterica serovar typhimurium was more susceptible to killing by heat, and grew poorly under conditions of high osmolarity. The wild-type and otsA mutant strains showed similar abilities to colonise spleen tissues after oral dosing of mice. These findings suggest that the otsBA gene products play a role in environmental survival, but not in virulence, of S. enterica serovar typhimurium.

The immunogenicity and protective efficacy of overlapping regions of the protective antigen (PA) polypeptide, cloned and expressed as glutathione S -transferase fusion proteins, have been assessed. Results show that protection can be attributed to individual domains and imply that it is domain 4 which contains the dominant protective epitopes of PA.

This study was supported by a core grant to P.T. from the GIS, an A-STAR research institute. The sequencing of the Burkholderia pseudomallei strains was supported by Wellcome Trust grant 098051 to J.P. Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. Publisher PDF

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis . Glycoconjugated LPS generated significantly higher antibody titres compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. From the Clinical Editor Burkholderia mallei is associated with multi-drug resistance, high mortality and potentials for weaponization through aerosol inhalation. The authors of this study present gold nanoparticles (AuNPs) functionalized with a glycoconjugate vaccine against this Gram negative bacterium demonstrating promising results in a murine model even with the aerosolized form of B. Mallei.

A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.

Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied., IMPORTANCE Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.

Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduc- tion in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed sig- nificantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

Melioidosis is a severe infectious disease caused byBurkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed ofB. pseudomalleilipopolysaccharide and theHcfragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge withB. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.

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 exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.

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.

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.

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

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.

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°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 .

Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively. Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare. The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B. pseudomallei and a strain of B. mallei which were suscep- tible to both antimicrobials in vitro. Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days post- challenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge. Mice were challenged ip with B. pseudomallei 4845 and hamsters were chal- lenged ip with B. mallei 23344. Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD). Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B. pseudomallei to 4 � 10 6 cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu. The results for doxycycline were similar. Ciprofloxacin prophylaxis raised the MLD of B. mallei 23344 to 4.6 � 10 5 cfu compared with 4 cfu in untreated controls. Immediate therapy raised the MLD to 7.0 � 10 4 cfu and therapy raised the MLD to 1.6 � 10 3 cfu. All regimens of doxycycline protected hamsters against challenges of up to 2 � 10 7 cfu. Despite using a susceptible strain of B. pseudomallei, neither antimicrobial was effective when used therapeutically. The timely administration of either antimicrobial, however, was effective in pre- venting symptomatic infection. Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4-5 weeks after challenge.

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.

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.

Clostridium perfringens isolates are currently classified into one of five biotypes on the basis of the differential production of α-, β-, ε- and ι-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 β2-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 β-toxin was present in 50% of the isolates genotyped. A significant association between C. perfringens possessing the β2-toxin gene and diarrhoea in piglets was identified, suggesting that β2-toxin may play a key role in the pathogenesis of the disease.

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.

SUMMARY 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 μg 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 μg F1 + 10 μg V to 0.01 μg F1 + 0.01 μg V in a two-dose regimen. For animals immunized at the 1-μg 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 105 colony-forming units (CFU) virulent Y. pestis at this time point. However, immunization with 5 μg or more of each subunit was required to achieve protection against challenge with 107 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

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.

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.

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 β1-linked galactosyl residues in glycosphingolipids is the minimum determinant required for binding of the pH 6 antigen.

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 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