10 research outputs found

    Chorismate synthase from Staphylococcus aureus

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    The aroC gene encoding chorismate synthase and the ndk gene encoding nucleoside diphosphate kinase were cloned from Staphylococcus aureus by complementation of the Aro- phenotype of the aroC E. coli strain, GLW40. Two partial open reading frames (ORFs) encoding 3-dehydroquinate synthase and a protein which had similarity with the putative gerCC gene product from B. subtilis were also cloned. In S. aureus the aroC gene is likely to form the first gene in an operon which includes the aroB and aroA genes. It has been demonstrated in this study that the ndk genes and the gerCAgerCBgerCC genes in S. aureus are situated upstream from the aroCaroB genes; this gene organisation has also been observed in B. subtilis. The S. aureus aroC gene was expressed from the T7 promoter on plasmid pTB361. This expression system resulted in the accumulation of very high levels of soluble S. aureus chorismate synthase and facilitated the purification of the enzyme to near homogeneity, producing 100mg of enzyme from 13g of cells. No detectable immunological crossreactivity was observed between S. aureus chorismate synthase and antibodies raised against E. coli chorismate synthase. This contrasts with other chorismate synthases and indicates that there are structural differences between the chorismate synthases from S. aureus and E. coli. S. aureus chorismate synthase was determined to be a homotetramer using gel filtration and chemical crosslinking. The pH optimum determined for S. aureus chorismate synthase was found to be non-symmetrical in MOPS buffer with an optimum of activity around pH 7.0. The apparent Km for EPSP of the S. aureus enzyme was calculated to be 12.7muM and the apparent Km for FMN was calculated to be 4.8muM. The apparent Km value for FMN for the S. aureus enzyme is two orders of magnitude greater compared to other chorismate synthases, excluding the B. subtilis enzyme. S. aureus chorismate synthase was investigated using pre-steady state kinetics and a flavin intermediate was observed during turnover with a difference spectrum resembling that obtained with the E. coli enzyme. The spectral characteristics of the S. aureus flavin intermediate were different, however, with respect to its maxima, minima and its overall shape. The rate of decay (6.5s-1) of the intermediate was eight times slower than that observed for the E. coli enzyme (52s-1) and this compares well with a 7 fold lower Vmax. Tyrosine 121 of S. aureus chorismate synthase was changed to phenylalanine or alanine using site-directed mutagenesis. The conversion to alanine resulted in a loss of activity while the phenylalanine mutant retained 10% of wild-type activity

    The biosynthesis of phylloquinone(vitamin K1) in higher plants

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    Phylloquinone is a compound present in all plants serving as cofactor for photosystem I mediated electron transport during photosynthesis. This work reports on the identification and analysis of several Arabidopsis thaliana phylloquinone absence (pha) and isochorismate synthase (ics) mutants impaired in the biosynthesis of PhQ (vitamin K1). Besides the complete lack of PhQ, these plants show a typical phenotype characterized by seedling lethality, photosynthetic defects specifically related to impaired photosystem I accumulation/activity to 5-15% of wild-type levels and partial recovery of 15% PhQ content and 50-70% PSI accumulation/activity after feeding with the metabolic precursor of vitamin K1, 1,4-dihydroxy-2-naphthoate. Map-based localization of the mutated allele in the pha plants identified a new gene, called PHYLLO. It consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of the photosynthetic phylloquinone in cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes still reside as polycistronic units in plastomes of red algae and in eubacterial chromosomes strongly suggests that PHYLLO derived from an operon present in the proto-organelle precursor of all plastids. The principle architecture of the PHYLLO locus is conserved in the nuclear genomes of plants and the green alga Chlamydomonas reinhardtii, indicating that selective forces have been acting to maintain the cluster structure in the form of a gene fusion, presumably as an adaptation of an multifunctional association of four enzymatic activities already pre-existing in the chloroplast. In line with this finding, the data present in this work suggest that the PHYLLO composite product is part of a metabolon for the biosynthesis of phylloquinone. The menF module of PHYLLO in Chlamydomonas, encoding the isochorismate synthase activity, is full-length, whereas in higher plants this module surprisingly lacks the functional 3’ part, uncovering a recent gene splitting event during evolution. Such a gene fission event, which resulted in inactivation of the encoded ICS enzymatic activity from PHYLLO, must have been preceded by establishment of a second functional copy of the menF gene. Accordingly, double-knockouts of the ICS1 and ICS2 genes in Arabidopsis analysed during this work, were unable to synthesize PhQ, demonstrating that the activity of the menF module of PHYLLO has been replaced after the splitting of the 3’-region by at least one more ICS gene present in genomes of higher plants. The fact that ICS1 is also required for salicylic acid biosynthesis in Arabidopsis, establishes a metabolic link between photosynthesis and systemic acquired resistance. Therefore, gene fusion, duplication and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants. Despite the essential function of PhQ for PSI stability and plant viability, analyses of ics heterozygous knockout plants, as well as complementation of the pha mutants by NA feeding and transgenic forms of PHYLLO demonstrate that the bulk of cellular phylloquinone is not associated with photosystem I, opening the possibility for additional functions of vitamin K1 in plant cell membranes

    A multidimensional approach towards studying recurrent Clostridium difficile infection

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    Clostridium difficile infection (CDI) is an infection of the gastrointestinal tract causing symptoms ranging from mild diarrhoea to life-threatening toxic megacolon. Between 10-30% of patients suffer a recurrent episode (rCDI) after an initial episode. Some patients develop multiple recurrent episodes, leading to unpleasant cycles of disease and antimicrobial therapy. This thesis utilises a multidimensional approach to study rCDI. In Chapter 2, previously generated clinical data is used to assess the effect of treatment delay on two outcomes; diarrhoeal duration and risk of recurrence. It was hypothesised that delays initiating treatment result in increased symptom duration and recurrence risk. Logistic regression models highlighted treatment delay has no significant effect on diarrhoeal duration or recurrence risk. The only significant variable associated with risk of recurrence was previous CDI (P<0.001). These findings suggest clinicians should not be overly concerned by treatment delays in mild/moderate CDI. In Chapter 3, the germination and thermotolerance properties of five strains of C. difficile spores were investigated. In the nosocomial environment spores may be reingested by the patient, germinate and initiate fulminant disease. Additionally, spores can persist in the gastrointestinal tract and germinate in response to stimulatory cues. C. difficile spore recovery was optimised by using variety of media and supplements. The ribotype (RT) 078 strain germinated more efficiently in the absence of additional supplementation. RT 027/078 strains were more thermotolerant. Intrinsic differences in spore germination characteristics between clades could facilitate the increased ability of some strains to cause rCDI. In Chapter 4, an in vitro gut model was used to simulate rCDI. Previous research has characterised changes in the microbiota that occur in response to antibiotics. In this study a metaproteomic approach was utilised to study the overarching metabolic processes occurring during simulated rCDI. Although dysbiosis was evident, the metaproteome remained fairly constant throughout simulated infection

    Mode of Action Study of Para-aminosalicylic Acid and Structure, Function and Inhibitor Study of the Isocitrate Dehydrogenase-2 in Mycobacterium tuberculosis

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    Tuberculosis (TB) killed 1.5 million people and rivaled AIDS, becoming the leading cause of death from infectious disease in 2014. The prevalence of multidrug resistant TB has intensified the current therapeutic procedure, making it urgent to find novel anti-tubercular agents and to come up with solutions to retard the emergence of the drug resistance. This dissertation focuses on the identification of drug targets, the exploration of drug resistance mechanisms, and the identification of novel inhibitors. In the first part, the mechanism of action of the classic anti-tubercular drug, para-aminosalicylic acid (PAS), was explored through genetic, cell viability and molecular modeling studies. Dihydrofolate reductase (DHFR) was identified to be the putative intracellular target of PAS. In addition, the molecular mechanism of PAS resistance was intensively investigated for the clinically relevant Rv2671 up-regulation mutant. Biochemical assays showed that Rv2671 exhibited a low DHFR activity with a high Km for the substrate, 7, 8-dihydrofolate. X-ray crystal structure of the Rv2671 in complex with NADP+ and tetrahydrofolate (THF) further confirmed the structural similarity between Rv2671 and DHFR. These studies together suggested that PAS resistance of this mutant is derived from the ability to complement the DHFR activity with the high level of Rv2671. The second part of this dissertation details the characteristics of Mycobacterium tuberculosis isocitrate dehydrogenase-2 (Mtb IDH2). The kinetic study of Mtb IDH2 suggested that it catalyzes an ordered sequential reaction by binding NADP+ first. X-ray crystal structure revealed the fairly conserved active site and dissimilar overall structure compared to human IDHs (HIDHs), suggesting a potential for drug selectivity. A screening of known inhibitors of mutant HIDHs and a high-throughput screening of Mtb whole cell active compounds were further implemented to identify inhibitors for Mtb IDH2. Two compounds from the screenings exhibited IC50s below 10 μM. The enzyme structure and the modest potency inhibitors of Mtb IDH2 can serve as viable starting points for the follow-up inhibitor development of Mtb IDH2

    Mode of Action Study of Para-aminosalicylic Acid and Structure, Function and Inhibitor Study of the Isocitrate Dehydrogenase-2 in Mycobacterium tuberculosis

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    Tuberculosis (TB) killed 1.5 million people and rivaled AIDS, becoming the leading cause of death from infectious disease in 2014. The prevalence of multidrug resistant TB has intensified the current therapeutic procedure, making it urgent to find novel anti-tubercular agents and to come up with solutions to retard the emergence of the drug resistance. This dissertation focuses on the identification of drug targets, the exploration of drug resistance mechanisms, and the identification of novel inhibitors. In the first part, the mechanism of action of the classic anti-tubercular drug, para-aminosalicylic acid (PAS), was explored through genetic, cell viability and molecular modeling studies. Dihydrofolate reductase (DHFR) was identified to be the putative intracellular target of PAS. In addition, the molecular mechanism of PAS resistance was intensively investigated for the clinically relevant Rv2671 up-regulation mutant. Biochemical assays showed that Rv2671 exhibited a low DHFR activity with a high Km for the substrate, 7, 8-dihydrofolate. X-ray crystal structure of the Rv2671 in complex with NADP+ and tetrahydrofolate (THF) further confirmed the structural similarity between Rv2671 and DHFR. These studies together suggested that PAS resistance of this mutant is derived from the ability to complement the DHFR activity with the high level of Rv2671. The second part of this dissertation details the characteristics of Mycobacterium tuberculosis isocitrate dehydrogenase-2 (Mtb IDH2). The kinetic study of Mtb IDH2 suggested that it catalyzes an ordered sequential reaction by binding NADP+ first. X-ray crystal structure revealed the fairly conserved active site and dissimilar overall structure compared to human IDHs (HIDHs), suggesting a potential for drug selectivity. A screening of known inhibitors of mutant HIDHs and a high-throughput screening of Mtb whole cell active compounds were further implemented to identify inhibitors for Mtb IDH2. Two compounds from the screenings exhibited IC50s below 10 μM. The enzyme structure and the modest potency inhibitors of Mtb IDH2 can serve as viable starting points for the follow-up inhibitor development of Mtb IDH2

    The ecophysiology of nitrite-oxidizing bacteria in the genus Nitrospira

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    Ein Kernprozess des biogeochemischen Stickstoffzyklus in natürlichen Lebensräumen und technischen Anlagen (z. B. Kläranlagen) ist die Nitrifikation. Dieser aerobe Prozess untergliedert sich in zwei Stufen, der oxidativen Umwandlung von Ammoniak zu Nitrit bzw. von Nitrit zu Nitrat. Zwei verschiedene funktionelle Gruppen chemolithotropher Mikroorganismen sind dabei katalytisch an der Stoffumsetzung beteiligt: die Ammoniak oxidierenden Bakterien (AOB) bzw. Archaeen (AOA) und die Nitrit oxidierenden Bakterien (NOB). Ein Hauptaugenmerk in dieser Doktorarbeit wurde auf NOB der Gattung Nitrospira gelegt. Diese Gruppe von Bakterien ist maßgeblich an der Stickstoffeleminierung in Kläranlagen beteiligt und ist darüber hinaus weit verbreitet in den unterschiedlichsten aquatischen und terrestrischen Lebensräumen. Bisher ist wenig bekannt über die Ökophysiologie dieser meist unkultivierbaren NOB. In dieser Arbeit konnte zum ersten Mal auf Metagenomsequenzen dieser Bakteriengruppe zurückgegriffen werden und zusammen mit dem Einsatz verschiedener molekularbiologischer Techniken erschlossen sich bis dato unbekannte ökophysiologische Charakteristika dieser NOB. Der gekoppelte Einsatz von Fluoreszenz in situ Hybridisierung (FISH) und quantitativer Bildanalyse ergab Hinweise für eine Nischendifferenzierung zweier Nitrospira Populationen in einem Biofilm aufgrund ihrer Präferenz für verschiedene Nitritkonzentrationen. Weitere physiologische Adaptationen dieser NOB ergaben sich aus Metagenomanalysen. Nitrospira besitzt das detoxifizierende Enzym Chloritdismutase. Es ist gut möglich, dass Nitrospira den Abbau von Chlor-Komponenten mit der Nitrifizierung an kontaminierten Standorten koppelt. Die Genomdaten lieferten darüber hinaus erste Einblicke in das bisher unbekannte Nitrit-oxidierende System dieser NOB. Diese Doktorarbeit erweitert unser bisheriges Verständnis über die Ökophysiologie Nitrit-oxidierender Nitrospiren. Der gekoppelte Einsatz neuer molekularer Methoden mit ersten Metagenomdaten ermöglichte eine detaillierte Beschreibung dieser langsam wachsenden, meist unkultivierbaren NOB.Nitrification is a key process of the biogeochemical nitrogen-cycle in natural and engineered habitats. The two steps of aerobic nitrification, the oxidation of ammonia to nitrite and subsequently from nitrite to nitrate, are catalysed by two functional groups of chemolithotrophic prokaryotes: the ammonia-oxidizing bacteria (AOB) and archaea (AOA), and the nitrite-oxidizing bacteria (NOB). This thesis focused on the genus Nitrospira, one of the important but yet less intensively studied groups of NOB. Nitrospira-like bacteria are the dominant NOB in wastewater treatment plants and the most diverse known NOB in nature, being widespread in various aquatic and terrestrial habitats. However, ecophysiological and genomic data of these mostly uncultured NOB are either scarce or lacking. Molecular and, for the first time metagenomic data retrieved in the course of this thesis brought forward novel ecophysiological traits of these NOB. Fluorescence in situ hybridization and quantitative image analysis provided indication of a different spatial arrangement of members of two Nitrospira sub-populations in a biofilm suggesting a niche differentiation of the coexisting NOB with respect to their preferred concentrations of nitrite. Metagenome analyses revealed further ecophysiological adaptations of these NOB. Nitrospira harbours a gene encoding the detoxifying enzyme chlorite dismutase. Therefore, Nitrospira might link the bioremediation of chloroxo compounds with nitrogen turnover at contaminated sites. Moreover, the metagenomic data uncovered a unique nitrite-oxidizing system in this NOB. This thesis extends our knowledge of the ecophysiology of nitrite-oxidizing Nitrospira-like bacteria. Combining novel molecular methods with metagenomic data allowed in-depth characterization of this slow-growing, mostly uncultured NOB

    Prophylaxis of disease caused by bacterial pathogens of man

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    This thesis reports research undertaken which will lead to improved pretreatments and therapies for disease caused by Clostridium perfringens, Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. C. perfringens is thought to be the most widely distributed bacterial pathogen and is the most important Clostridial species associated with enteric disease in domesticated animals. During warfare C. perfringens has been a significant causes of mortality. Between 1 and 10% of wounded personnel developed gas gangrene during the 1st and 2nd world wars. The ability of the bacterium to cause a range of diseases is due largely to the differential production of toxins. The first reported cloning and nucleotide sequencing of three of the four major toxins (α, β and ε-toxins) is documented in this thesis. The regulation of expression of α-toxin in C. perfringens has been investigated and methods for the expression of recombinant proteins in E. coli have been devised This information has been used to develop improved PCR-based diagnostic tests, and to investigate structure-function relationships. A high resolution crystal structure of a-toxin (phospholipase C) is reported. Using molecular and biophysical techniques, the functions of the two domains of the protein have been determined. Residues that play roles in the interaction of the toxin with host cell membranes have been identified using site-directed mutagenesis. This work has also provided a major insight into the structures and functions of related phospholipases C (the zincmetallophospholipases C) from other bacterial pathogens. This pioneering work with α-toxin is recognised by invitations to write reviews and book chapters on this subject and on bacterial phospholipases C. C. perfringens β-toxin has been shown to be related to pore forming toxins such as Staphylococcus aureus α-toxin. This finding suggests, for the first time, the mode of action of β-toxin. The interaction of C. perfringens ε-toxin with host cells has been investigated and progress made in identifying the cell-surface receptor for the toxin. Genetically engineered toxoids have been devised which induce high-level protection against α and ε-toxins. These vaccines are currently being developed by industry for veterinary use. Similar approaches have been used to devise a recombinant vaccine against Clostridium botulinum toxin F. The wider applications of toxins as therapeutics have also been investigated, and a novel cancer drug delivery system based on targeted lysis of drug-containing liposomes by α-toxin has been devised and patented. F. tularensis is the etiological agent of tularemia, a disease of man that is found in most countries in the Northern hemisphere and most frequently in Scandinavia, N. America, Japan and N. Russia. In this thesis the efficacy of antibiotics for the prevention and treatment of experimental tularemia is documented. Two surface antigens (lipopolysaccharide and FopA) have been evaluated as sub-unit vaccines. Of these, lipopolysaccharide shows potential as a protective antigen. However, because of the paucity of information available on this bacterium, a wider approach to vaccine development, involving the determination of the genome sequence of a fully virulent strain of F. tularensis has been undertaken. A preliminary analysis of the genome sequence is reported here, which has allowed the identification of targets for the development of a rationally attenuated mutant for use as a live vaccine. Y. pestis is generally recognised to have caused three major pandemics of disease, and credible estimates indicate that together these resulted in 200 million deaths. WHO figures indicate that there is a continuing public health problem from plague, especially in Africa, Asia and South America. In this thesis existing vaccines and antibiotics have been evaluated for the prevention and treatment of plague and found to have limitations. A number of approaches to the development of an improved vaccine have been investigated including rationally attenuated strains of the bacterium and isolated surface antigens. A sub-unit vaccine against plague has been devised based on recombinant forms of the F1- and V-antigens. This vaccine provides high level protection against both bubonic and pneumonic plague. This recombinant sub-unit vaccine has been patented and is currently in phase I clinical trials in man. This vaccine has been formulated for single oral or intranasal delivery, using microencapsulated or Salmonella-based delivery systems. Methods for enhancing the stability and efficacy of these vaccines have been investigated. Reviews on plague and plague vaccines have been written, confirming the status of the author as a world leader in this field. The work to devise an improved vaccine has also provided insight into the molecular basis of pathogencity of Y. pestis. A phoP / phoQ regulatory system has been discovered in the bacterium, which plays a key role in survival of the bacterium within macrophages. The V-antigen has been shown to be surface located to play a key role in the translocation of effector proteins into host cells. The biogenesis of the F1-capsular antigen has been investigated at a genetic and biophysical level. In order to underpin future work with this pathogen, the genome sequence is currently being determined. This work has already provided major new insights into the evolution of this pathogen. B. pseudomallei (formerly Pseudomonas pseudomallei) is found primarily in S. E. Asia, N. Australia and other tropical areas of the world. Melioidosis has recently appeared in temperate zones, including mainland France and the UK possible as a consequence of increased international travel. Acute disease can be treated with antibiotics but the bacterium can persist in the host and subsequent disease episodes can occur. In this thesis ciprofloxacin and doxycyline have been are evaluated and shown to have significant limitations for the treatment of melioidodis. In the longer term there is a requirement for an effective vaccine against melioidosis, and work is reported here to devise the genetic tools which will be necessary for the genetic manipulation of the bacterium, with a view towards the identification of virulence determinants

    Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

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    peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts
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