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Professor Ian Charles

Biography

Ian Charles is a visiting professor within the University of Technology Sydney, Faculty of Science. He has over 30 years’ experience in academic and commercial research. His academic career has included being the previous head of ithree institute (UTS), and he’s a founding member of The Wolfson Institute for Biomedical Research at University College London, one the UK’s first institutes of translational medicine. He has also worked in the pharmaceutical industry at Glaxo Wellcome, and has been founder and CSO of biotech companies in the area of infectious disease, including Arrow Therapeutics, sold to AstraZeneca, and Auspherix a venture capital backed company founded in 2013 at UTS. His current research interests include infectious diseases as well as the microbiome and its impact on health and wellbeing. Ian is now the director of the Institute of Food Research, Norwich, UK, and he continues to collaborate with scientists within the ithree institute at UTS.

Image of Ian Charles
Visiting Professor, Faculty of Science
Core Member, ithree - Institute of Infection, Immunity and Innovation
PhD
 

Chapters

Charles, I.G., Foxwell, N. & Chubb, A. 1997, 'Cloning and expression of human eNOS and nNOS using the baculovirus-insect cell system' in Titheradge, M.A. (ed), Methods in molecular biology: nitric oxide protocols, Humana Press, England, pp. 51-60.
To date, the cDNAs and genes encoding three distinct forms of the enzyme nitric oxide synthase (NOS) have been described (reviewed in ref. 1). The family of NOS enzymes generate nitric oxide (NO) from arginine, producing citrulline as a co-product and requires the presence of heme, tetrahydrobiopterin (BH4), nicotinamide adenine dinucleotide phosphate (NADPH), calmodulin (CaM), flavin mononucleotide (FMN), and flavine adenine nucleotide (FAD) (2). The NO generated by the family of NO synthases has been found to play an important role in many biological processes involved in health and disease including neurotransmission, the mediation of vasodilation, and host defense.
Charles, I.G., Chubb, A., Keeling, J., Moncada, S. & Riveros-Moreno, V. 1994, 'High expression of rat brain NO synthase' in Moncada, S., Feelish, M., Busse, R. & Higgs, E.A. (eds), The biology of nitric oxide, Portland Press Ltd, United Kingdom, pp. 18-21.
Charles, I.G., Page, M., Scorer, C., Foxwell, N., Knowles, R., Holmes, L.S., Chubb, A., Palmer, R.M. & Moncada, S. 1994, 'Expression of human inducible NO synthase cDNA in a baculovirus expression system' in Moncada, S., Feelish, M., Busse, R. & Higgs, E.A. (eds), The biology of nitric oxide, Portland Press Ltd, United Kingdom, pp. 316-320.
Weiner, C.P., Lizasoain, I., Bayliss, S.A., Knowles, R., Charles, I.G. & Moncada, S. 1994, 'Calcium dependent nitic oxide dynthase, their induction by sex hormones' in Moncada, S., Feelish, M., Busse, R. & Higgs, E.A. (eds), The biology of nitric oxide, Portland Press Ltd, United Kingdom, pp. 9-13.
Xu, W., Charles, I.G., Moncada, S., Gorman, P., Liu, L. & Emsom, P.C. 1994, 'Chromosomal assignment of the inducible NOS gene and endothelium NOS gene to human chromosome 17p11-q11 and chromosome 7 respectively' in Moncada, S., Feelish, M., Busse, R. & Higgs, E.A. (eds), The biology of nitric oxide, Portland Press Ltd, United Kingdom, pp. 121-125.
Chatfield, S.N., Fairweather, N., Tite, J., Charles, I.G., Roberts, M., Posada, M., Strugnell, R.A. & Dougan, G. 1991, 'The development of genetically defined live bacterial vaccines' in Wadstrom, T., Helena Mäkelä, P., Svennerholm, A.M. & Wolf-Watz, H. (eds), Molecular pathogenesis of gastrointestinal infections, Plenum Press, New York, pp. 279-285.

Conferences

Chatfield, S.N., Fairweather, N., Roberts, M., Tite, J., Charles, I.G. & Dougan, G. 1990, 'Rationally attenuated Salmonella strains as live oral vaccines', Proceedings of the 5th European Congress on Biotechnology, European Congress on Biotechnology, Copenhagen, pp. 182-185.
Fairweather, N., Makoff, A.J., Oxer, M.D., Dougan, G., Ballantine, S.P., Roberts, M. & Charles, I.G. 1990, 'P.69 pertactin, a protective antigen from Bordetella pertussis: high level expression and purification from Escherichia coli and protective properties', Proceedings of the Sixth International Symposium on Pertussis, Dept. of Health and Human Services, Maryland.
Charles, I.G., Li, L.J., Strugnell, R.A., Dougan, G., Novotny, P., Heron, I., Au Jensen, M., Manclark, C., Brennan, M. & Fairweather, N. 1990, 'Repeat sequence motifs constitute the immunodominant regions of the P.69 pertactin from Bordetella pertussis: comparison with repeat sequences from B. parapertussis and B. bronchiseptica', Proceedings of the Sixth International Symposium on Pertussis, Dept. of Health and Human Services, Maryland, pp. 136-140.

Journal articles

Gloag, E.S., Elbadawi, C., Zachreson, C.J., Aharonovich, I., Toth, M., Charles, I.G., Turnbull, L. & Whitchurch, C.B. 2017, 'Micro-Patterned Surfaces That Exploit Stigmergy to Inhibit Biofilm Expansion.', Frontiers in Microbiology, vol. 7, pp. 1-10.
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Twitching motility is a mode of surface translocation that is mediated by the extension and retraction of type IV pili and which, depending on the conditions, enables migration of individual cells or can manifest as a complex multicellular collective behavior that leads to biofilm expansion. When twitching motility occurs at the interface of an abiotic surface and solidified nutrient media, it can lead to the emergence of extensive self-organized patterns of interconnected trails that form as a consequence of the actively migrating bacteria forging a furrow network in the substratum beneath the expanding biofilm. These furrows appear to direct bacterial movements much in the same way that roads and footpaths coordinate motor vehicle and human pedestrian traffic. Self-organizing systems such as these can be accounted for by the concept of stigmergy which describes self-organization that emerges through indirect communication via persistent signals within the environment. Many bacterial communities are able to actively migrate across solid and semi-solid surfaces through complex multicellular collective behaviors such as twitching motility and flagella-mediated swarming motility. Here, we have examined the potential of exploiting the stigmergic behavior of furrow-mediated trail following as a means of controlling bacterial biofilm expansion along abiotic surfaces. We found that incorporation of a series of parallel micro-fabricated furrows significantly impeded active biofilm expansion by Pseudomonas aeruginosa and Proteus vulgaris. We observed that in both cases bacterial movements tended to be directed along the furrows. We also observed that narrow furrows were most effective at disrupting biofilm expansion as they impeded the ability of cells to self-organize into multicellular assemblies required for escape from the furrows and migration into new territory. Our results suggest that the implementation of micro-fabricated furrows that exploit stigmergy may be a ...
Joss, T.V., Burke, C.M., Hudson, B.J., Darling, A.E., Forer, M., Alber, D.G., Charles, I.G. & Stow, N.W. 2016, 'Bacterial Communities Vary between Sinuses in Chronic Rhinosinusitis Patients.', Frontiers in microbiology, vol. 6, pp. 1532-1532.
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Chronic rhinosinusitis (CRS) is a common and potentially debilitating disease characterized by inflammation of the sinus mucosa for longer than 12 weeks. Bacterial colonization of the sinuses and its role in the pathogenesis of this disease is an ongoing area of research. Recent advances in culture-independent molecular techniques for bacterial identification have the potential to provide a more accurate and complete assessment of the sinus microbiome, however there is little concordance in results between studies, possibly due to differences in the sampling location and techniques. This study aimed to determine whether the microbial communities from one sinus could be considered representative of all sinuses, and examine differences between two commonly used methods for sample collection, swabs, and tissue biopsies. High-throughput DNA sequencing of the bacterial 16S rRNA gene was applied to both swab and tissue samples from multiple sinuses of 19 patients undergoing surgery for treatment of CRS. Results from swabs and tissue biopsies showed a high degree of similarity, indicating that swabbing is sufficient to recover the microbial community from the sinuses. Microbial communities from different sinuses within individual patients differed to varying degrees, demonstrating that it is possible for distinct microbiomes to exist simultaneously in different sinuses of the same patient. The sequencing results correlated well with culture-based pathogen identification conducted in parallel, although the culturing missed many species detected by sequencing. This finding has implications for future research into the sinus microbiome, which should take this heterogeneity into account by sampling patients from more than one sinus.
Chowdhury, P.R., DeMaere, M., Chapman, T., Worden, P., Charles, I.G., Darling, A.E. & Djordjevic, S.P. 2016, 'Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease', BMC MICROBIOLOGY, vol. 16.
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Roy Chowdhury, P., Scott, M., Worden, P., Huntington, P., Hudson, B., Karagiannis, T., Charles, I. & Djordjevic, S. 2016, 'Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa', OpenBiology, vol. 6.
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Turnbull, L., Toyofuku, M., Hynen, A.L., Kurosawa, M., Pessi, G., Petty, N.K., Osvath, S.R., Carcamo-Oyarce, G., Gloag, E.S., Shimoni, R., Omasits, U., Ito, S., Yap, X., Monahan, L.G., Cavaliere, R., Ahrens, C.H., Charles, I.G., Nomura, N., Eberl, L. & Whitchurch, C.B. 2016, 'Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms', NATURE COMMUNICATIONS, vol. 7.
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Wyrsch, E., Roy Chowdhury, P., Chapman, T.A., Charles, I.G., Hammond, J.M. & Djordjevic, S.P. 2016, 'Genomic Microbial Epidemiology Is Needed to Comprehend the Global Problem of Antibiotic Resistance and to Improve Pathogen Diagnosis', Frontiers in Microbiology, vol. 7, no. 843.
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Wyrsch, E., Roy Chowdhury, P., Chapman, T.A., Charles, I.G., Hammond, J.M. & Djordjevic, S.P. 2016, 'Genomic Microbial Epidemiology Is Needed to Comprehend the Global Problem of Antibiotic Resistance and to Improve Pathogen Diagnosis', Frontiers in Microbiology, vol. 7, no. 843.
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Labbate, M., Orata, F.D., Petty, N.K., Jayatilleke, N.D., King, W.L., Kirchberger, P.C., Allen, C., Mann, G., Mutreja, A., Thomson, N.R., Boucher, Y. & Charles, I.G. 2016, 'A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules.', Sci Rep, vol. 6, p. 36891.
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Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS's. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules.
Labbate, M., Islam, A., Monahan, L.G., Charles, I.G. & Stokes, H.W. 2015, 'A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage', Environmental Microbiology.
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Roy Chowdhury, P., Charles, I.G. & Djordjevic, S.P. 2015, 'A role for Tn6029 in the evolution of the complex antibiotic resistance gene loci in genomic island 3 in enteroaggregative hemorrhagic Escherichia coli O104:H4.', PloS one, vol. 10, no. 2, p. e0115781.
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In enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104 the complex antibiotic resistance gene loci (CRL) found in the region of divergence 1 (RD1) within E. coli genomic island 3 (GI3) contains blaTEM-1, strAB, sul2, tet(A)A, and dfrA7 genes encoding resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim respectively. The precise arrangement of antibiotic resistance genes and the role of mobile elements that drove the evolutionary events and created the CRL have not been investigated. We used a combination of bioinformatics and iterative BLASTn searches to determine the micro-evolutionary events that likely led to the formation of the CRL in GI3 using the closed genome sequences of EAHEC O104:H4 strains 2011C-3493 and 2009EL-2050 and high quality draft genomes of EAHEC E. coli O104:H4 isolates from sporadic cases not associated with the initial outbreak. Our analyses indicate that the CRL in GI3 evolved from a progenitor structure that contained an In2-derived class 1 integron in a Tn21/Tn1721 hybrid backbone. Within the hybrid backbone, a Tn6029-family transposon, identified here as Tn6029C abuts the sul1 gene in the 3'-Conserved Segment (-CS) of a class 1 integron generating a unique molecular signature that has only previously been observed in pASL01a, a small plasmid found in commensal E. coli in West Africa. From this common progenitor, independent IS26-mediated events created two novel transposons identified here as Tn6029D and Tn6222 in 2011C-3493 and 2009EL-2050 respectively. Analysis of RD1 within GI3 reveals IS26 has played a crucial role in the assembly of regions within the CRL.
Wyrsch, E., Roy Chowdhury, P., Abraham, S., Santos, J., Darling, A.E., Charles, I.G., Chapman, T.A. & Djordjevic, S.P. 2015, 'Comparative genomic analysis of a multiple antimicrobial resistant enterotoxigenic E. coli O157 lineage from Australian pigs.', BMC genomics, vol. 16, pp. 165-165.
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BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) are a major economic threat to pig production globally, with serogroups O8, O9, O45, O101, O138, O139, O141, O149 and O157 implicated as the leading diarrhoeal pathogens affecting pigs below four weeks of age. A multiple antimicrobial resistant ETEC O157 (O157 SvETEC) representative of O157 isolates from a pig farm in New South Wales, Australia that experienced repeated bouts of pre- and post-weaning diarrhoea resulting in multiple fatalities was characterized here. Enterohaemorrhagic E. coli (EHEC) O157:H7 cause both sporadic and widespread outbreaks of foodborne disease, predominantly have a ruminant origin and belong to the ST11 clonal complex. Here, for the first time, we conducted comparative genomic analyses of two epidemiologically-unrelated porcine, disease-causing ETEC O157; E. coli O157 SvETEC and E. coli O157:K88 734/3, and examined their phylogenetic relationship with EHEC O157:H7. RESULTS: O157 SvETEC and O157:K88 734/3 belong to a novel sequence type (ST4245) that comprises part of the ST23 complex and are genetically distinct from EHEC O157. Comparative phylogenetic analysis using PhyloSift shows that E. coli O157 SvETEC and E. coli O157:K88 734/3 group into a single clade and are most similar to the extraintestinal avian pathogenic Escherichia coli (APEC) isolate O78 that clusters within the ST23 complex. Genome content was highly similar between E. coli O157 SvETEC, O157:K88 734/3 and APEC O78, with variability predominantly limited to laterally acquired elements, including prophages, plasmids and antimicrobial resistance gene loci. Putative ETEC virulence factors, including the toxins STb and LT and the K88 (F4) adhesin, were conserved between O157 SvETEC and O157:K88 734/3. The O157 SvETEC isolate also encoded the heat stable enterotoxin STa and a second allele of STb, whilst a prophage within O157:K88 734/3 encoded the serum survival gene bor. Both isolates harbor a large repertoire of antibi...
Darling, A.E., Worden, P.J., Chapman, T., Roy Chowdhury, P., Charles, I.G. & Djordjevic, S.P. 2014, 'The genome of Clostridium difficile 5.3', Gut Pathogens, vol. 6, no. 4.
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Background Clostridium difficile is the leading cause of infectious diarrhea in humans and responsible for large outbreaks of enteritis in neonatal pigs in both North America and Europe. Disease caused by C. difficile typically occurs during antibiotic therapy and its emergence over the past 40 years is linked with the widespread use of broad-spectrum antibiotics in both human and veterinary medicine. Results We sequenced the genome of Clostridium difficile 5.3 using the Illumina Nextera XT and MiSeq technologies. Assembly of the sequence data reconstructed a 4,009,318 bp genome in 27 scaffolds with an N50 of 786 kbp. The genome has extensive similarity to other sequenced C. difficile genomes, but also has several genes that are potentially related to virulence and pathogenicity that are not present in the reference C. difficile strain. Conclusion Genome sequencing of human and animal isolates is needed to understand the molecular events driving the emergence of C. difficile as a gastrointestinal pathogen of humans and food animals and to better define its zoonotic potential.
Monahan, L.G., Turnbull, L., Osvath, S.R., Birch, D., Charles, I.G. & Whitchurch, C.B. 2014, 'Rapid conversion of Pseudomonas aeruginosa to a spherical cell morphotype facilitates tolerance to carbapenems and penicillins but increases susceptibility to antimicrobial peptides', Antimicrobial Agents and Chemotherapy, vol. 58, no. 4, pp. 1956-1962.
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The Gram negative human pathogen Pseudomonas aeruginosa is able to tolerate high concentrations of -lactam antibiotics. Despite inhibiting the growth of the organism, these cell wall-targeting drugs exhibit remarkably little bactericidal activity. However, the mechanisms underlying -lactam tolerance are currently unclear. Here we show that P. aeruginosa undergoes a rapid en masse transition from normal rod shaped cells to viable, cell wall defective spherical cells when treated with -lactams from the widely used carbapenem and penicillin classes. When the antibiotic is removed, the entire population of spherical cells quickly converts back to the normal bacillary form. Our results demonstrate that these rapid population-wide cell morphotype transitions function as a strategy to survive antibiotic exposure. Taking advantage of these findings, we have developed a novel approach to efficiently kill P. aeruginosa by using carbapenem treatment to induce en masse transition to the spherical cell morphotype and then exploiting the relative fragility and sensitivity of these cells to killing by antimicrobial peptides (AMPs) that are relatively inactive against P. aeruginosa bacillary cells. This approach could broaden the repertoire of antimicrobial compounds used to treat P. aeruginosa and serve as a basis for developing new therapeutics to combat bacterial infections.
Monahan, L.G., Hajduk, I.V., Blaber, S.P., Charles, I.G. & Harry, E.J. 2014, 'Coordinating Bacterial Cell Division with Nutrient Availability: a Role for Glycolysis', MBIO, vol. 5, no. 3.
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Islam, M.A., Labbate, M., Djordjevic, S.P., Alam, M., Darling, A.E., Melvold, J.A., Holmes, A.J., Johura, F.T., Cravioto, A., Charles, I.G. & Stokes, H. 2013, 'Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic', Open Biology, vol. 3, p. 120181.
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Of the 200þ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a nonendemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in nonO1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.
Gloag, E.S., Turnbull, L., Huang, A., Vallotton, P., Wang, H., Nolan, L.M., Mililli, L., Hunt, C., Lu, J., Osvath, S.R., Monahan, L.G., Cavaliere, R., Charles, I.G., Wand, M., Gee, M., Ranganathan, P. & Whitchurch, C.B. 2013, 'Self-organization of bacterial biofilms is facilitated by extracellular DNA', Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 28, pp. 11541-11546.
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Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the bulldozer aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA
Chaudhuri, R., Morgan, E., Peters, S.E., Pleasance, S., Hudson, D., Davies, H., Wang, J., Van Diemen, P., Buckley, A., Bowen, A., Pullinger, G., Turner, D., Langridge, G., Turner, A.K., Parkhill, J., Charles, I.G., Maskell, D. & Stevens, M.P. 2013, 'Comprehensive Assignment Of Roles For Salmonella Typhimurium Genes In Intestinal Colonization Of Food-producing Animals', PLoS Genetics, vol. 9, no. 4, pp. 1-11.
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Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotypegenotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens.
Nichols, C.E., Lamb, H.K., Thompson, P., El Omari, K., Lockyer, M., Charles, I.G., Hawkins, A.R. & Stammers, D.K. 2013, 'Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD-YeaZ-YjeE complex', Protein Science, vol. 22, no. 5, pp. 628-640.
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YgjD from COG0533 is amongst a small group of highly conserved proteins present in all three domains of life. Various roles and biochemical functions (including sialoprotease and endonuclease activities) have been ascribed to YgjD and orthologs, the most recent, however, is involvement in the post transcriptional modification of certain tRNAs by formation of N6-threonyl-adenosine (t6A) at position 37. In bacteria, YgjD is essential and along with YeaZ, YjeE, and YrdC has been shown to be 'necessary and sufficient' for the tRNA modification. To further define interactions and possible roles for some of this set of proteins we have undertaken structural and biochemical studies. We show that formation of the previously reported heterodimer of YgjD-YeaZ involves ordering of the C-terminal region of YeaZ which extends along the surface of YgjD in the crystal structure. ATP?S or AMP is observed in YgjD while no nucleotide is bound on YeaZ. ITC experiments reveal previously unreported binary and ternary complexes which can be nucleotide dependent. The stoichiometry of the YeaZ-YgjD complex is 1:1 with a K(D) of 0.3 µM. YgjD and YjeE interact only in the presence of ATP, while YjeE binds to YgjD-YeaZ in the presence of ATP or ADP with a K(D) of 6 µM. YgjD doesn't bind the precursors of t6A, threonine, and bicarbonate. These results show a more complex set of interactions than previously thought, which may have a regulatory role. The understanding gained should help in deriving inhibitors of these essential proteins that might have potential as antibacterial drugs.
Stamp, A.L., Owen, P., El Omari, K., Lockyer, M., Lamb, H.K., Charles, I.G., Hawkins, A.R. & Stammers, D.K. 2011, 'Crystallographic and microcalorimetric analyses reveal the structural basis for high arginine specificity in the Salmonella enterica serovar Typhimurium periplasmic binding protein STM4351', Proteins: Structure, Function and Genetics, vol. 79, no. 7, pp. 2352-2357.
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Periplasmic binding proteins (PBPs) are crucial transport system components of prokaryotes, required for bacterial growth and survival. PBPs are also used by Gram negative bacteria for small molecule active transport via ATP-binding cassette (ABC) transporters or in signal transduction by inner membrane chemotaxis regulators. Many ligands for PBPs have been identified including amino acids, peptides, vitamins, metal ions, anions, etc. Eight distinct clusters of PBPs have been assigned based on ligand specificity together with amino acid sequence alignments.
Stamp, A., Owen, P., El Omari, K., Nichols, C., Lockyer, M., Lamb, H., Charles, I.G., Hawkins, A. & Stammers, D. 2010, 'Structural and functional characterization of Salmonella enterica serovar typhimurium YcbL: An unusual type II glyoxalase', Protein Science, vol. 19, no. 10, pp. 1897-1905.
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YcbL has been annotated as either a metallo-β-lactamase or glyoxalase II (GLX2), both members of the zinc metallohydrolase superfamily, that contains many enzymes with a diverse range of activities. Here, we report crystallographic and biochemical data for Salmonella enterica serovar Typhimurium YcbL that establishes it as GLX2, which differs in certain structural and functional properties compared with previously known examples. These features include the insertion of an α-helix after residue 87 in YcbL and truncation of the C-terminal domain, which leads to the loss of some recognition determinants for the glutathione substrate. Despite these changes, YcbL has robust GLX2 activity. A further difference is that the YcbL structure contains only a single bound metal ion rather than the dual site normally observed for GLX2s. Activity assays in the presence of various metal ions indicate an increase in activity above basal levels in the presence of manganous and ferrous ions. Thus, YcbL represents a novel member of the GLX2 family.
Peters, S.E., Paterson, G.K., Bandularatne, E.S., Northen, H., Pleasance, S.J., Willers, C., Wang, J., Foote, A.K., Constantino-Casas, F., Scase, T.J., Blacklaws, B.A., Bryant, C.E., Mastroeni, P., Charles, I.G. & Maskell, D. 2010, 'Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.', Infection and Immunity, vol. 78, no. 1, pp. 326-336.
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In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4 and CD8 T cells and B lymphocytes in the spleen and reduced infiltration by CD11b cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.
Langridge, G., Phan, M.D., Turner, D.J., Perkins, T., Parts, L., Haase, J., Charles, I.G., Maskell, D., Peters, S.E., Dougan, G., Wain, J., Parkhill, J. & Turner, A.K. 2009, 'Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.', Genome Research, vol. 19, no. 12, pp. 2308-2316.
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Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.
Chaudhuri, R., Peters, S., Pleasance, S., Northen, H., Willers, C., Paterson, G., Cone, D., Allen, A., Owen, P., Shalom, G., Stekel, D., Charles, I.G. & Maskell, D. 2009, 'Comprehensive Identification Of Salmonella Enterica Serovar Typhimurium Genes Required For Infection Of Balb/C Mice', Plos Pathogens, vol. 6, no. 7, pp. 1-13.
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Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains tha
Chaudhuri, R.R., Allen, A.G., Owen, P.J., Shalom, G., Stone, K., Harrison, M., Burgis, T.A., Lockyer, M., Garcia-Lara, J., Foster, S.J., Pleasance, S.J., Peters, S.E., Maskell, D. & Charles, I.G. 2009, 'Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH)', BMC Genomics, vol. 10, no. 291, pp. 1-18.
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Background In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. Results We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. Conclusion We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.
Langridge, G., Phan, M.D., Turner, D.J., Perkins, T., Parts, L., Haase, J., Charles, I.G., Maskell, D., Peters, S.E., Dougan, G., Wain, J., Parkhill, J. & Turner, A.K. 2009, 'Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.', Genome Research, vol. 19, no. 12, pp. 2308-2316.
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Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.
Lamb, H., Thompson, P., Elliott, C.J., Charles, I.G., Richards, J., Lockyer, M., Watkins, N., Nichols, C., Stammers, D., Bagshaw, C., Cooper, A. & Hawkins, A. 2007, 'Functional Analysis Of The Gtpases Enga And Yhbz Encoded By Salmonella Typhimurium', Protein Science, vol. 16, no. 11, pp. 2391-2402.
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The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtA(E) of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome matu
Nichols, C., Lamb, H., Lockyer, M., Charles, I.G., Pyne, S.G., Hawkins, A. & Stammers, D. 2007, 'Characterization Of Salmonella Typhimurium Yegs, A Putative Lipid Kinase Homologous To Eukaryotic Sphingosine And Diacylglycerol Kinases', Proteins-Structure Function And Bioinformatics, vol. 68, no. 1, pp. 13-25.
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Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzyme
Nichols, C.E., Johnson, C., Lamb, H.K., Lockyer, M., Charles, I.G., Hawkins, A.R. & Stammers, D.K. 2007, 'Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium', Acta Crystallographica Section F-Structural Biology And Crystallization Communications, vol. 63, no. 11, pp. 922-928.
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The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.
El Omari, K., Dhaliwal, B., Lockyer, M., Charles, I.G., Hawkins, A. & Stammers, D. 2006, 'Structure Of Staphylococcus Aureus Guanylate Monophosphate Kinase', Acta Crystallographica Section F-Structural Biology..., vol. 62, no. 10, pp. 949-953.
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Nucleotide monophosphate kinases (NMPKs) are potential antimicrobial drug targets owing to their role in supplying DNA and RNA precursors. The present work reports the crystal structure of Staphylococcus aureus guanylate monophosphate kinase (SaGMK) at 1
Dhaliwal, B., Ren, J., Lockyer, M., Charles, I.G., Hawkins, A. & Stammers, D. 2006, 'Structure Of Staphylococcus Aureus Cytidine Monophosphate Kinase In Complex With Cytidine 5 '-Monophosphate', Acta Crystallographica Section F-Structural Biology And Crystallization Communications, vol. 62, no. 8, pp. 710-715.
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The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 50-monophosphate (CMP) has been determined at 2.3 angstrom resolution. The active site reveals novel features when compared with two orthologues o
Mo, E., Peters, S., Willers, C., Maskell, D. & Charles, I.G. 2006, 'Single, Double And Triple Mutants Of Salmonella Enterica Serovar Typhimurium Degp (Htra), Degq (Hhoa) And Degs (Hhob) Have Diverse Phenotypes On Exposure To Elevated Temperature And Their Growth In Vivo Is Attenuated To Different Extents', Microbial Pathogenesis, vol. 41, no. 4-5, pp. 174-182.
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DegP (HtrA) is a well-studied protease involved in survival of bacteria under stress conditions in vitro and in vivo. There are two paralogues of DegP in the Salmonella enterica serovar Typhimurium genome, DegQ and DegS. In order to understand more about
Nichols, C., Johnson, C., Lockyer, M., Charles, I.G., Lamb, H., Hawkins, A. & Stammers, D. 2006, 'Structural Characterization of Salmonella typhimurium Yeaz, an M22 O-Sialoglycoprotein Endopeptidase Homolog', Proteins-Structure Function And Bioinformatics, vol. 64, no. 1, pp. 111-123.
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The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gep. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide. binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.
Lamb, H.K., Mee, C., Xu, W., Liu, L., Blond, S., Cooper, A., Charles, I.G. & Hawkins, A.R. 2006, 'The Affinity of a Major Ca2+ Binding Site on GRP78 Is Differentially Enhanced by ADP and ATP', Journal of Biological Chemistry, vol. 281, no. 13, pp. 8796-8805.
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GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered ?11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered ?40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.
Xu, W., Charles, I.G. & Moncada, S. 2005, 'Nitric oxide: orchestrating hypoxia regulation through mitochondrial respiration and the endoplasmic reticulum stress response', Cell Research, vol. 15, no. 13, pp. 63-65.
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Mitochondria have long been considered to be the powerhouse of the living cell, generating energy in the form of the molecule ATP via the process of oxidative phosphorylation. In the past 20 years, it has been recognised that they also play an important role in the implementation of apoptosis, or programmed cell death. More recently it has become evident that mitochondria also participate in the orchestration of cellular defence responses. At physiological concentrations, the gaseous molecule nitric oxide (NO) inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen. This interaction underlies the mitochondrial actions of NO, which range from the physiological regulation of cell respiration, through mitochondrial signalling, to the development of "metabolic hypoxia" a situation in which, although oxygen is available, the cell is unable to utilise it.
Xu, W., Liu, L., Charles, I.G. & Moncada, S. 2004, 'Nitric Oxide Induces Coupling Of Mitochondrial Signalling With The Endoplasmic Reticulum Stress Response', Nature Cell Biology, vol. 6, no. 11, pp. 1129-1134.
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Nitric oxide ( NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen(1-3). This action of NO has both physiological and pathophysiological consequences. Here we report that endo
Park, A., Lamb, H., Nichols, C., Moore, J., Brown, K.R., Cooper, A., Charles, I.G., Stammers, D. & Hawkins, A. 2004, 'Biophysical And Kinetic Analysis Of Wild-Type And Site-Directed Mutants Of The Isolated And Native Dehydroquinate Synthase Domain Of The Arom Protein', Protein Science, vol. 13, no. 8, pp. 2108-2119.
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Dehydroquinate synthase (DHQS) is the N-terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes. DHQS converts 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate i
Nichols, C.E., Ren, J., Leslie, K., Dhaliwal, B., Lockyer, M., Charles, I.G., Hawkins, A.R. & Stammers, D.K. 2004, 'Comparison of ligand-induced conformational changes and domain closure mechanisms, between prokaryotic and eukaryotic dehydroquinate synthase', Journal of Molecular Biology, vol. 343, no. 3, pp. 533-546.
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Dehydroquinate synthase (DHQS) is a potential target for the development of novel broad-spectrum antimicrobial drugs, active against both prokaryotes and lower eukaryotes. Structures have been reported for Aspergillus nidulans DHQS (AnDHQS) in complexes with a range of ligands. Analysis of these AnDHQS structures showed that a large-scale domain movement occurs during the normal catalytic cycle, with a complex series of structural elements propagating substrate binding-induced conformational changes away from the active site to distal locations. Compared to corresponding fungal enzymes, DHQS from bacterial species are both mono-functional and significantly smaller. We have therefore determined the structure of Staphylococcus aureus DHQS (SaDHQS) in five liganded states, allowing comparison of ligand-induced conformational changes and mechanisms of domain closure between fungal and bacterial enzymes. This comparative analysis shows that substrate binding initiates a large-scale domain closure in both species' DHQS and that the active site stereochemistry, of the catalytically competent closed-form enzyme thus produced, is also highly conserved. However, comparison of AnDHQS and SaDHQS open-form structures, and analysis of the putative dynamic processes by which the transition to the closed-form states are made, shows a far lower degree of similarity, indicating a significant structural divergence. As a result, both the nature of the propagation of conformational change and the mechanical systems involved in this propagation are quite different between the DHQSs from the two species.
Dhaliwal, B., Nichols, C.E., Ren, J., Lockyer, M., Charles, I.G., Hawkins, A.R. & Stammers, D.K. 2004, 'Crystallographic studies of shikimate binding and induced conformational changes in Mycobacterium tuberculosis shikimate kinase', FEBS Letters, vol. 574, no. 1, pp. 49-54.
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The X-ray crystal structure of Mycobacterium tuberculosis shikimate kinase (SK) with bound shikimate and adenosine diphosphate (ADP) has been determined to a resolution of 2.15 Å. The binding of shikimate in a shikimate kinase crystal structure has not previously been reported. The substrate binds in a pocket lined with hydrophobic residues and interacts with several highly conserved charged residues including Asp34, Arg58, Glu61 and Arg136 which project into the cavity. Comparisons of our ternary SKADPshikimate complex with an earlier binary SKADP complex show that conformational changes occur on shikimate binding with the substrate-binding domain rotating by 10°. Detailed knowledge of shikimate binding is an important step in the design of inhibitors of SK, which have potential as novel anti-tuberculosis agents.
Spiers, A., Lamb, H., Cocklin, S., Wheeler, K., Budworth, J., Dodds, A., Pallen, M., Maskell, D., Charles, I.G. & Hawkins, A. 2002, 'Pdz Domains Facilitate Binding Of High Temperature Requirement Protease A (Htra) And Tail-Specific Protease (Tsp) To Heterologous Substrates Through Recognition Of The Small Stable Rna A (Ssra)-Encoded Peptide', Journal Of Biological Chemistry, vol. 277, no. 42, pp. 39443-39449.
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The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protei
Bird, L.E., Ren, J., Zhang, J., Foxwell, N., Hawkins, A.R., Charles, I.G. & Stammers, D.K. 2002, 'Crystal Structure of SANOS, a Bacterial Nitric Oxide Synthase Oxygenase Protein from Staphylococcus aureus', Structure, vol. 10, no. 12, pp. 1687-1696.
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Prokaryotic genes related to the oxygenase domain of mammalian nitric oxide synthases (NOSs) have recently been identified. Although they catalyze the same reaction as the eukaryotic NOS oxygenase domain, their biological function(s) are unknown. In order to explore rationally the biochemistry and evolution of the prokaryotic NOS family, we have determined the crystal structure of SANOS, from methicillin-resistant Staphylococcus aureus (MRSA), to 2.4 Å. Haem and S-ethylisothiourea (SEITU) are bound at the SANOS active site, while the intersubunit site, occupied by the redox cofactor tetrahydrobiopterin (H4B) in mammalian NOSs, has NAD+ bound in SANOS. In common with all bacterial NOSs, SANOS lacks the N-terminal extension responsible for stable dimerization in mammalian isoforms, but has alternative interactions to promote dimer formation.
Xu, W., Liu, L. & Charles, I.G. 2002, 'Microencapsulated iNOS-expressing cells cause tumor suppression in mice', Faseb Journal, vol. 16, pp. 213-215.
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Macrophages are capable of killing tumor cells by releasing nitric oxide (NO) and reactive nitrogen species after expression of the inducible nitric oxide synthase (iNOS) gene. We intend to develop a novel cell-based therapy for tumor inhibition using microencapsulated cells capable of expressing iNOS under control of a regulated promoter
Xu, W., Liu, L., Loizidou, M., Ahmed, M. & Charles, I.G. 2002, 'The role of nitric oxide in cancer', Cell Research, vol. 12, no. 311, p. 320.
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Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties. Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis by upregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53, poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease.
Khan, S.J., Strijbos, P., Everest, P., Moss, D.S., Stratford, R., Mastroeni, P., Allen, J., Servos, S., Charles, I.G., Dougan, G. & Maskell, D. 2001, 'Early Responses To Salmonella Typhimurium Infection In Mice Occur At Focal Lesions In Infected Organs', Microbial Pathogenesis, vol. 30, no. 1, pp. 29-38.
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Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid, with key roles for cytokines and possibly also inducible nitric oxide synthase (iNOS), in mediating host responses to infection. In this paper we demonstrat
Selwood, D.L., Brummell, D.G., Budworth, J., Burtin, G.E., Campbell, R.O., Chana, S.S., Charles, I.G., Fernandez, P.A., Glen, R.C., Goggin, M.C., Hobbs, A.J., Kling, M.R., Liu, Q., Madge, D.J., Meillerais, S., Powell, K.L., Reynolds, K., Spacey, G.D., Stables, J.N., Tatlock, M.A., Wheeler, K.A., Wishart, G. & Woo, C. 2001, 'Synthesis and Biological Evaluation of Novel Pyrazoles and Indazoles as Activators of the Nitric Oxide Receptor, Soluble Guanylate Cyclase', Journal Of Medicinal Chemistry, vol. 44, pp. 78-93.
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Database searching and compound screening identified !-benzyl-3-(3-dimethylaminopropyloxy). indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A ompr~hensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However' replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing)no significant inhibition of phosphodiesterases or nitric oxide synthases.
Xu, W., Humphries, S., Tomita, M., Okuyama, T., Matsuki, M., Burgner, D., Kwiatkowski, D., Liu, L. & Charles, I.G. 2000, 'Survey of the Allelic Frequency of a NOS2A Promoter Microsatellite in Human Populations: Assessment of the NOS2A Gene and Predisposition to Infectious Disease', Nitric Oxide-Biology And Chemistry, vol. 4, no. 4, pp. 379-383.
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Allelic frequencies of a (CCTTT) n pentanucleotide repeat in the NOS2A promoter region were determined in a total of 1393 unrelated individuals from five specific population groups in four continents: Africa, Europe, Asia, and the Caribbean. There were highly significant differences in allele frequencies between the ethnically diverse populations. The repeat variation may have implications for the selective pressure of malaria or other infectious diseases that may operate at the NOS2 locus.
Xu, W., Liu, L., Charles, I.G. & Smith, G.C. 2000, 'Nitric Oxide Upregulates Expression Of Dna-Pkcs To Protect Cells From Dna-Damaging Anti-Tumour Agents', Nature Cell Biology, vol. 2, no. 6, pp. 339-345.
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Nitric-oxide synthase (NOS) activity has been detected in many human tumours, although its function is unclear. Here we show that exposure of cells to nitric oxide (NO) results in a 4-5-fold increase in expression of the DNA-dependent protein-kinase cata
Emsley, P., Fotinou, C., Black, I., Fairweather, N., Charles, I.G., Watts, C., Hewitt, E. & Isaacs, N. 2000, 'The Structures Of The H-C Fragment Of Tetanus Toxin With Carbohydrate Subunit Complexes Provide Insight Into Ganglioside Binding', Journal Of Biological Chemistry, vol. 275, no. 12, pp. 8889-8894.
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The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, whic
Xu, W., Liu, L., Emson, P., Harrington, C., Mckeith, I., Perry, R.H., Morris, C. & Charles, I.G. 2000, 'The Ccttt Polymorphism In The Nos2A Gene Is Associated With Dementia With Lewy Bodies', Neuroreport, vol. 11, no. 2, pp. 297-299.
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We report the analysis of the allele distribution of a (CCTTT)n pentanucleotide repeat within the promoter region of the NOS2A gene in DNA samples from patients with autopsy confirmed Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) type. A s
Maria, J.S., Vallance, P., Charles, I.G. & Leiper, J.M. 1999, 'Identification of microbial dimethylarginine dimethylaminohydrolase enzymes', Molecular Microbiology, vol. 33, no. 6, pp. 1278-1279.
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Arginine metabolism is an important source of energy and nitrogen in matly microbes. In this report, we present the sequence and activity of a novel microbial enzyme located in certain arginine-handling operons. The enzyme is homologous to mammalian dimethylarginine dimethylaminohydrolases (DDAH) and metabolizes asymmetric methylarginines to citrulline.
Strijbos, P., Pratt, G., Khan, S.J., Charles, I.G. & Garthwaite, J. 1999, 'Molecular Characterization And In Situ Localization Of A Full-Length Cyclic Nucleotide-Gated Channel In Rat Brain', European Journal of Neuroscience, vol. 11, no. 12, pp. 4463-4467.
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Ion channels gated directly by cyclic nucleotides are required for the transduction of sensory signals in photoreceptor cells and olfactory cells. Cyclic nucleotide-gated (CNG) channels may also be expressed in the central nervous system because partial
Leiper, J., Maria, J., Chubb, A., Macallister, R., Charles, I.G., Whitley, G. & Vallance, P. 1999, 'Identification Of Two Human Dimethylarginine Dimethylaminohydrolases With Distinct Tissue Distributions And Homology With Microbial Arginine Deiminases', Biochemical Journal, vol. 343, no. 1, pp. 209-214.
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Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3.18). We have cloned human DDAH and identified and expressed
Baylis, S., Strijbos, P., Sandra, A., Russell, R., Rijhsinghani, A., Charles, I.G. & Weiner, C. 1999, 'Temporal Expression Of Inducible Nitric Oxide Synthase In Mouse And Human Placenta', Molecular Human Reproduction, vol. 5, no. 3, pp. 277-286.
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The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription-polymerase chain reaction (RT-PCR), Norther
Warpeha, K.M., Xu, W., Liu, L., Charles, I.G., Patterson, C.C., Ah-fat, F., Harding, S., Hart, P.M., Chakravarthy, U. & Hughes, A.E. 1999, 'Genotyping and functional analysis of a polymorphic (CCTTT)n repeat of NOS2A in diabetic retinopathy', Faseb Journal, vol. 13, pp. 1825-1832.
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Accumulating evidence shows that the severity and rapidity of onset of diabetic retinopathy are influenced by genetic factors. Expression of the nitric oxide synthases is altered in the retinal vasculature in the early stages of diabetic retinopathy. We analyzed the allele distribution of a polymorphic pentanucleotide repeat within the 5' upstream promoter region of the NOS2A gene in samples of diabetic patients. In diabetic patients from Northern Ireland, the 14-repeat allele of the NOS2A marker was significantly associated with the absence of diabetic retinopathy. Carriers of this repeat had 0.21-fold the relative risk of developing diabetic retinopathy than noncarriers of this allele. They also had significantly fewer renal and cardiovascular complications. The ability of differing numbers of (CCTTT)n pentanucleotide repeats to induce transcription of the NOS2A gene was analyzed using a luciferase reporter gene assay in transfected colonic carcinoma cells. Interleukin 1 (IL-1) induction was most effective in constructs carrying the 14-repeat allele. When cells were incubated in 25 mM glucose to mimic the diabetic state, IL-1 induction was inhibited in all cases, but to a significantly lesser extent with the 14-repeat allele. These unique properties of the 14-repeat allele may confer selective advantages in diabetic individuals, which may delay or prevent microvascular complications of diabetes.Warpeha, K. M., Xu, W., Liu, L., Charles, I. G., Patterson, C. C., Ah-Fat, F., Harding, S., Hart, P. M., Chakravarthy, U., Hughes, A. E. Functional analysis of the polymorphic (CCTTT)n locus of NOS2A in diabetic retinopathy.
Budworth, J., Meillerais, S., Charles, I.G. & Powell, K. 1999, 'Tissue Distribution of the Human Soluble Guanylate Cyclases', Biochemical and Biophysical Research Communication, vol. 263, no. 3, pp. 696-701.
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Soluble guanylate cyclase (sGC) is an important component of the NO signaling pathway. Human sGC isoforms ?1, ?2, and ?1 show differential mRNA tissue distributions. ?1 and ?1 are expressed in most tissues; however, the ?2 isoform shows a more restricted expression pattern with high levels in brain, placenta, spleen, and uterus only. Both ? subunits exist as multiple transcripts whereas ?1 exists as a single message. This study reports for the first time the tissue distribution of human sGC message and demonstrates that sGC isoforms are nonuniformly expressed which may be useful if the enzyme is to be exploited as a therapeutic target.
Bhagat, K., Hingorani, A.D., Palacios, M., Charles, I.G. & Vallance, P. 1999, 'Cytokine-induced venodilatation in humans in vivo: eNOS masquerading as iNOS', Cardiovascular Research, vol. 41, no. 3, pp. 754-764.
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Objective: Venodilatation is a feature of endotoxaemia and sepsis. We have tested directly the hypothesis that three cytokines (IL-1?, TNF? and IL-6) generated during endotoxaemia affect venous tone in humans in vivo by increasing NO generation and explored whether the NO comes from the iNOS or eNOS isoform. Design and intervention: Cytokines were given into a superficial vein in very low doses sufficient only to produce changes in the study vessel. The effects of cytokines on the response to noradrenaline were examined. Results: IL-1? increased basal NO-induced dilatation in the study vein, and this was sufficient to attenuate the constrictor response to exogenous noradrenaline or sympathetic stimulation. The effects were maximal at 6 h and both NG-monomethyl-l-arginine and aminoguanidine caused significant reversal of the IL-1? effects. However, no induction of iNOS mRNA was detected in the tissue samples. Instead, mRNA encoding eNOS and GTP cyclohydrolase-1 was detected in all vessels. Conclusion: The simplest explanation of these results is that IL-1? induces expression of GTP cyclohydrolase-1 which leads to increased generation of BH4 and activation of eNOS. This study identifies IL-1? as a key cytokine causing physiologically significant venodilatation in humans by increasing NO generation and suggests that this can occur even in the absence of iNOS expression.
Khan, S.J., Everest, P., Servos, S., Foxwell, N., Zahringer, U., Brade, H., Rietschel, E., Dougan, G., Charles, I.G. & Maskell, D. 1998, 'A Lethal Role For Lipid A In Salmonella Infections', Molecular Microbiology, vol. 29, no. 2, pp. 571-579.
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Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been availa
Vallance, P. & Charles, I.G. 1998, 'Nitric oxide as an antimicrobial agent: does NO always mean NO?', Gut: an international journal of gastroenterology & hepatology, vol. 42, pp. 313-314.
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Nitric oxide (NO) synthesised from l-arginine subserves multiple physiological functions in the cardiovascular, respiratory, gastrointestinal, genitourinary, and central and peripheral nervous systems.1 But synthesis of NO also contributes to host defence and seems to have cytostatic and cytotoxic effects against certain pathogens, and even against host cells themselves.1 How is this double act achieved? What determines the switch from physiological mediator to lethal gas and how is bacterial killing achieved?
Burgner, D., Xu, W., Rockett, K., Gravenor, M.E., Charles, I.G., Hill, A.L. & Kwiatkowski, D. 1998, 'Inducible Nitric Oxide Synthase Polymorphism And Fatal Cerebral Malaria', The Lancet, vol. 352, no. 9135, pp. 1193-1194.
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There has been much speculation about the part played by nitric oxide (NO) in malaria, both as an antiparasitic agent and as a potential cause of neurosuppression leading to cerebral malaria.[1] and [2] Investigation is hampered by difficulty in estimating in-vivo production of NO, but genetic studies provide a potential means of examining the relation between NO production and disease outcome. A report from Gabon suggests that a single nucleotide polymorphism in the inducible nitric oxide synthase (NOS2) promoter is associated with protection from all forms of severe malaria, including susceptibility to reinfection.3 We present data from The Gambia suggesting that a similar region of the NOS2 gene encodes a susceptibility determinant for fatal cerebral malaria.
Moilanen, E., Moilanen, T., Knowles, R., Charles, I.G., Kadoya, Y., Alsaffar, N., Revell, P. & Moncada, S. 1997, 'Nitric Oxide Synthase Is Expressed In Human Macrophages During Foreign Body Inflammation', American Journal Of Pathology, vol. 150, no. 3, pp. 881-887.
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Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants lead
Xu, W., Liu, L., Emson, P., Harrington, C. & Charles, I.G. 1997, 'Evolution Of A Homopurine-Homopyrimidine Pentanucleotide Repeat Sequence Upstream Of The Human Inducible Nitric Oxide Synthase Gene', Gene, vol. 204, no. 1-2, pp. 165-170.
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We have identified a highly polymorphic pentanucleotide repeat (CCTTT)n within the 5'-putative promoter region of the human inducible nitric oxide synthase gene (iNOS, NOS2). Using a pair of specific primers derived from the human iNOS gene, we have also
Boyhan, A., Smith, D.B., Charles, I.G., Saqi, M. & Lowe, P. 1997, 'Delineation Of The Arginine- And Tetrahydrobiopterin-Binding Sites Of Neuronal Nitric Oxide Synthase', Biochemical Journal, vol. 323, no. 1, pp. 131-139.
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Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (tetrahydro-biopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists
Cubberley, R., Alderton, W., Boyhan, A., Charles, I.G., Lowe, P. & Old, R. 1997, 'Cysteine-200 Of Human Inducible Nitric Oxide Synthase Is Essential For Dimerization Of Haem Domains And For Binding Of Haem, Nitroarginine And Tetrahydrobiopterin', Biochemical Journal, vol. 323, no. 1, pp. 141-146.
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Nitric oxide synthase (EC 1.14.13.39) is a homodimer. Limited proteolysis has previously shown that it consists of two major domains. The C-terminal or reductase domain binds FMN, FAD and NADPH. The N-terminal or oxygenase domain is known to bind arginin
Robinson, N., Westmore, K., Martin, J.F., Emson, P. & Charles, I.G. 1997, 'Inducible Nitric Oxide Synthase Gene Transcription And Protein Activity In The Rat Heart During Endotoxaemia', BIOCHEMICAL AND BIOPHYSICAL RESEARCH C..., vol. 231, no. 1, pp. 211-216.
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Septicaemia leads to an impairment of myocardial contractility in animals and humans. Cytokines released during endotoxaemia are capable of increasing inducible nitric oxide synthase (iNOS) expression in vitro in myocytes, endothelial cells and macrophag
Smith, R.E., Robinson, N.M., McPeake, J.R., Baylis, S.A., Charles, I.G., Heaton, N.D., Moncada, S., Williams, R. & Martin, J.F. 1997, 'Induction and Role of NO Synthase in Hypotensive Hepatic Failure', Arteriosclerosis Thrombosis And Vascular Biology, vol. 17, no. 11, pp. 3079-3082.
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Nitric oxide (NO) plays an important role in the physiological and pathophysiological control of the vascular system. NO is synthesized by isoforms of the enzyme NO synthase (NOS). Hepatic failure is complicated by hypotension, low systemic vascular resistance, and resistance to vasoconstrictor drugs. The potential role of NO in these abnormalities was investigated by using in vitro pharmacological interventions on hepatic arteries obtained from both donor and recipient patients at the time of liver transplantation. The presence of NOS mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) with primers designed from human endothelial NOS (eNOS) and inducible NOS (iNOS) cDNA sequences. Arteries from patients with hepatic failure had an impaired constrictor response to phenylephrine compared with those of donor arteries. The constrictor effect of phenylephrine was potentiated by NG-monomethyl-L-arginine, an inhibitor of NOS, which had no effect in donor control arteries. RT-PCR identified human eNOS mRNA in donor and recipient arteries and human iNOS mRNA in recipient arteries only. Induction of NOS in the vasculature with subsequent NO-induced vasodilatation may therefore contribute to the hemodynamic abnormalities observed in hepatic failure and potentially in other pathologies associated with endotoxemia. Whether selective inhibitors of iNOS will improve hemodynamic control or clinical outcome in these conditions requires further study.
Everest, P., Li, J., Douce, G., Charles, I.G., Deazavedo, J., Chatfield, S., Dougan, G. & Roberts, M. 1996, 'Role Of The Bordetella Pertussis P.69/Pertactin Protein And The P.69/Pertactin Rgd Motif In The Adherence To And Invasion Of Mammalian Cells', Microbiology-uk, vol. 142, no. 11, pp. 3261-3268.
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The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located pm gene were significantly more invasive th
Lowe, P., Smith, D.B., Stammers, D., Riverosmoreno, V., Moncada, S., Charles, I.G. & Boyhan, A. 1996, 'Identification Of The Domains Of Neuronal Nitric Oxide Synthase By Limited Proteolysis', Biochemical Journal, vol. 314, no. 1, pp. 55-62.
Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-bind
Emsley, P., Charles, I.G., Fairweather, N. & Isaacs, N. 1996, 'Structure Of Bordetella Pertussis Virulence Factor P.69 Pertactin', Nature, vol. 381, no. 6577, pp. 90-92.
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A NEW generation of whooping-cough vaccines contain P.69 pertactin, a surface-exposed domain of an outer membrane protein expressed by the virulent bacterium Bordetella pertussis(1-3). This protein is a virulence factor that mediates adhesion to target m
Xu, W., Charles, I.G., Liu, L., Moncada, S. & Emson, P. 1996, 'Molecular Cloning and Structural Organization of the Human Inducible Nitric Oxide Synthase Gene (NOS2)', Biochemical and Biophysical Research Communication, vol. 219, no. 3, pp. 784-788.
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Previously, we reported the isolation and molecular cloning of human inducible nitric oxide synthase gene (NOS2) sequences from human chromosome 17 cosmid libraries. Here we describe the further characterization and sequencing of the NOS2 gene. The genomic structure of the NOS2 gene was determined from two overlapping cosmid clones, namely, pcos4A and pcos20. The NOS2 open reading frame is encoded by 27 exons, with translation initiation and termination in exon 2 and exon 27, respectively. These results differ from the previously reported organization of the iNOS gene, where 26 exons were reported for the genomic structure of NOS2.
Charles, I.G., Scorer, C., Moro, M.A., Fernandez, C., Chubb, A., Dawson, J., Foxwell, N., Knowles, R.G. & Bayliss, S.A. 1996, 'The expression of human nitric oxide synthase isozymes', Methods in Enzymology, vol. 268, pp. 449-460.
Wei, X., Charles, I.G., Smith, A.C., Ure, J., Feng, C., Huang, F.Y., Xu, D.X., Muller, W.J., Moncada, S. & Liew, F. 1995, 'Altered Immune-Responses In Mice Lacking Inducible Nitric-Oxide Synthase', Nature, vol. 375, no. 653, pp. 408-411.
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NITRIC oxide (NO) is important in many biological functions(1-5). It is generated from L-arginine by the enzyme NO synthase (NOS), The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantit
Jenkins, D., Charles, I.G., Thomsen, L., Moss, D.S., Holmes, L., Baylis, S., Rhodes, P., Westmore, K., Emson, P. & Moncada, S. 1995, 'Roles Of Nitric-Oxide In Tumor-Growth', Proceedings Of The National Academy Of Sciences ..., vol. 92, no. 1, pp. 4392-4396.
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A subclone of the human colon adenocarcinoma cell line DLD-1, which grew reproducibly as subcutaneous tumors in nude mice, was isolated. Such cells, when engineered to generate nitric oxide (NO) continuously, grew more slowly in vitro than the wild-type
Riveros-Moreno, V., Heffernan, B., Torres, B., Chubb, A., Charles, I.G. & Moncada, S. 1995, 'Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase', European Journal Of Biochemistry, vol. 230, no. 1, pp. 52-57.
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We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 ?g/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol min<sup>-1</sup> mg<sup>-1</sup>, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol min<sup>-1</sup> mg<sup>-1</sup>) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H<sub>4</sub>biopterin, 0.32, with a specific activity of 1500 nmol min<sup>-1</sup> mg<sup>-1</sup>. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis
Norris, P.J., Charles, I.G., Scorer, C.A. & Emson, P.C. 1995, 'Studies on the localization and expression of nitric oxide synthase using histochemical techniques', Journal of Molecular Histology, vol. 27, no. 10, pp. 745-756.
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This review provides an update on the variety of histochemical techniques available for the cellular localization and expression of nitric oxide synthase in formalin-fixed tissue sections. The techniques of immunohistochemistry and NADPH-diaphorase histochemistry are discussed and the suitability of various types of probes and reporters which are useful for in situ detection of nitric oxide synthase mRNA expression are assessed. Figures are also included which illustrate the techniques described and protocols for in situ hybridization and NADPH-diaphorase histochemistry.
Robinson, N.M., Smith, R.E., McPeake, J.R., Bayliss, S.A., Charles, I.G., Heaton, N.D., Williams, R., Martin, J.F. & Moncada, S. 1995, 'Detection and pathological role of inducible nitric oxide synthase in man', Circulation, vol. 92, pp. 2960-2960.
Moss, D.W., Wei, X., Liew, F.Y., Moncada, S. & Charles, I.G. 1995, 'Enzymatic characterisation of recombinant murine inducible nitric oxide synthase', European Journal of Pharmacology, vol. 289, pp. 41-48.
A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus-insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and K(m) values for these two substrates. The nitric oxide synthase antagonists N-guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme.
Xu, W., Charles, I.G., Liu, L., Koni, P.A., Moncada, S. & Emson, P. 1995, 'Molecular genetic analysis of the duplication of human inducible nitric oxide synthase (NOS2) sequences', Biochemical and Biophysical Research Communication, vol. 212, no. 2, pp. 466-472.
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In previous studies, we found multiple copies of inducible nitric oxide synthase gene (NOS2)-like sequences in the human genome and mapped them to the pericentric region of chromosome 17. Here, we describe the cloning and sequencing of exon22 regions from three of these NOS2-like sequences. We have also mapped another NOS2-like sequence to human chromosome 14. Since there are multiple NOS2-like sequences present in the human genome, we have also carried out Zoo Blot hybridisation analysis using a NOS2 cDNA probe. Our data suggest that duplication of NOS2-like sequences occurred very recently in primate evolution.
Jenkins, D., Charles, I.G., Baylis, S., Lelchuk, R., Radomski, M. & Moncada, S. 1994, 'Human Colon-Cancer Cell-Lines Show A Diverse Pattern Of Nitric-Oxide Synthase Gene-Expression And Nitric-Oxide Generation', British Journal Of Cancer, vol. 70, no. 5, pp. 847-849.
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A panel of human colonic adenocarcinoma cell lines was examined both for expression of mRNAs of the nitric oxide synthase (NOS) gene family and for evidence of enzymic activity based on citrulline and nitrite (NO2-) formation. Reverse transcription-polym
Weiner, C., Lizasoain, I., Baylis, S., Knowles, R., Charles, I.G. & Moncada, S. 1994, 'Induction Of Calcium-Dependent Nitric-Oxide Synthases By Sex-Hormones', Proceedings Of The National Academy Of Sciences ..., vol. 91, no. 11, pp. 5212-5216.
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We have examined the effects of pregnancy and sex hormones on calcium-dependent and calcium-independent nitric oxide synthases (NOSs) in the guinea pig. Pregnancy (near term) caused a >4-fold increase in the activity of calcium-dependent NOS in the uteri
Charles, I.G., Fairweather, N., Pickard, D., Beesley, J., Anderson, R., Dougan, G. & Roberts, M. 1994, 'Expression Of The Bordetella-Pertussis P.69 Pertactin Adhesin In Escherichia-Coli - Fate Of The Carboxy-Terminal Domain', Microbiology-uk, vol. 140, no. 12, pp. 3301-3308.
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The mature pertactin protein (P.69) of Bordetella pertussis can be isolated from the bacterial cell surface as a polypeptide with an apparent molecular mass of 69 000 Da as determined by sodium dodecyl sulphate gel electrophoresis. However the open readi
Garvey, E., Tuttle, J., Covington, K., Merrill, B., Wood, E., Baylis, S. & Charles, I.G. 1994, 'Purification And Characterization Of The Constitutive Nitric-Oxide Synthase From Human Placenta', Archives Of Biochemistry And Biophysics, vol. 311, no. 2, pp. 235-241.
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Human endothelial nitric oxide synthase (NOS) mRNA was detected in human placenta. In contrast, mRNAs for human neuronal NOS or for human inducible NOS were not detected in placenta. Subsequently, NOS was purified over 3800-fold from placental extract to
Xu, W., Charles, I.G., Moncada, S., Gorman, P., Sheer, D., Liu, L. & Emson, P.C. 1994, 'Mapping of the genes encoding human inducible and endothelial nitric oxide synthase (NOS2 and NOS3) to the pericentric region of chromosome 17 and to chromosome 7, respectively', Genomics, vol. 21, no. 2, pp. 419-422.
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Nitric oxide (NO) is an important molecular messenger regulating the functions of a wide variety of cells and tissues. NO is synthesized from Image -arginine by a variety of isoforms of the enzyme nitric oxide synthase (NOS). We have used Southern blotting analysis on DNAs obtained from a panel of human-rodent hybrid cell lines to map the gene encoding the inducible NOS (NOS2) to chromosome 17cen-17q11 and the gene encoding the endothelial form of NOS (NOS3) to chromosome 7. Fluorescence in situ hybridization using a NOS2 probe gave several signals in the 17p11-q11 pericentromeric region
Makoff, A.J., Charles, I.G. & Fairweather, N. 1994, 'Recombinant antigens as components of a Diptheria Tetanus Pertussis vaccine', Bioprocess Technology, vol. 19, pp. 205-231.
Mock, B.A., Krall, M.M., Byrd, L.G., Chin, H., Howard-Barton, C., Charles, I.G., Liew, F.Y. & Blackwell, J. 1994, 'The inducible form of nitric oxide synthase (Nos-2) isolated from murine macrophage maps near the Nude mutation on mouse chromosome 11', European Journal of Immunogenetics, vol. 21, pp. 231-238.
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Gourley, D.G., Coggins, J.R., Isaacs, N.W., Moore, J.D., Charles, I.G. & Hawkins, A.R. 1994, 'Crystallization of a type II dehydroquinase from Mycobacterium tuberculosis', Journal of Molecular Biology, vol. 241, no. 3, pp. 488-491.
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A type II 3-dehydroquinase from Mycobacterium tuberculosis has been crystallized in the presence of 6% polyethyleneglycol 6000. Data from these crystals have been collected to a resolution of 22 &Aring; on a rotating anode X-ray source. The space group has been determined as F23 with unit cell dimensions of a = B = c = 1278 &Aring;. There is one molecule in the asymmetric unit
Emsley, P., McDermott, G., Charles, I.G., Fairweather, N. & Isaacs, N.W. 1994, 'Crystallographic characterization of pertactin, a membrane-associated protein from Bordetella pertussis', Journal of Molecular Biology, vol. 235, no. 2, pp. 772-773.
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Pertactin, a membrane-associated protein of Bordetella pertussis, has been crystallized in the presence of 28% ammonium sulphate. The space group is P 6322 with cell dimensions a = b = 1782 &Aring; and c = 1068 &Aring;. The crystals diffract to 33 &Aring; using a rotating anode source and are suitable for an X-ray structure determination. Assuming one molecule in the asymmetric unit, 70% of the cell is occupied by solvent.
Charles, I.G., Rodgers, B., Musgrave, S., Peakman, T., Chubb, A., Fairweather, N., Dougan, G. & Roberts, M. 1993, 'Expression Of P.69/Pertactin From Bordetella-Pertussis In A Baculovirus/Insect Cell Expression System - Protective Properties Of The Recomnbinant Protein', Research in Microbiology, vol. 144, no. 9, pp. 681-690.
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The surface antigen P.69/pertactin of Bordetella pertussis has been expressed using the polyhedron promoter of baculovirus in cultured insect cells. Either full-length or truncated pm DNA was used to express P.69 pertactin. The full-length gene gave rise
Moore, J., Hawkins, A., Charles, I.G., Deka, R., Coggins, J., Cooper, A., Kelly, S. & Price, N. 1993, 'Characterization Of The Type-I Dehydroquinase From Salmonella-Typhi', Biochemical Journal, vol. 295, no. 1, pp. 277-285.
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The type I dehydroquinase from the human pathogen Salmonella typhi was overexpressed in an Escherichia coli host and purified to homogeneity. The S. typhi enzyme was characterized in terms of its kinetic parameters, important active-site residues, therma
Brett, S., Mazurov, A., Charles, I.G. & Tite, J. 1993, 'The invasin protein of Yersinia spp. provides costimulatory activity to human T-cells through interaction with beta-1 integrins', European Journal Of Immunology, vol. 23, no. 7, pp. 1608-1614.
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The invasin proteins of Yersinia spp. are outer membrane proteins which are involved in the penetration of these bacteria into mammalian cells (Cell 1990. 60: 861). Invasin binds to several different beta1 integrins with extremely high affinity, the inte
Hawkins, A.R., Lamb, H.K., Moore, J.D., Charles, I.G. & Roberts, C.F. 1993, 'The pre-chorismate (shikimate) and quinate pathways in filamentous fungi : theoretical and practical aspects', Journal of General Microbiology, vol. 139, no. 12, pp. 2891-2899.
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The detailed chemistry and enzymology of the shikimate pathway have been extensively discussed in recent excellent reviews (Pittard, 1987 ; Bentley, 1990 ; Braus, 1991). This short review will therefore focus ( a ) on the web of interconnecting evolutionary and structural relationships within and between the enzymes and regulatory proteins that constitute the pre-chorismate (shikimate) and the related quinate pathways, and ( b ) the possibility of pathway engineering to divert major new carbon flux from the quinate pathway into the shikimate pathway.
Charles, I.G., Palmer, R.M., Hickery, M.S., Bayliss, M.T., Chubb, A., Hall, V.S., Moss, D.W. & Moncada, S. 1993, 'Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte', Proceedings of the National Academy of Sciences, vol. 90, no. NA, pp. 11419-11423.
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Incubation of buman articular cbondrocytes with interleukin III results in the time-dependent expression of nitric oxide (NO) synthase. We report bere the isolatIon of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO ceUs transfected with a plasmid harboring Ibis eDNA clone expressed NO syntbase activity that was inhibited by some L-arginine analogues. Tbe deduced amino acid sequence of the human chondrocyte inducible NO synthase sbows 51 % identity and 68% simUarity witb the endotheUaI NO synthase and 54% identity and 70% sbnllarity with tbe neuronal NO synthase. The simUarity (88%) between tbe buman cbondrocyte NO synthase eDNA sequence and that reported for the murine macropbage suggests tbat tbe inducible class of enzyme -is conserved between different cell types and across species.
Palmer, R.M., Hickery, M.S., Charles, I.G., Moncada, S. & Bayliss, M.T. 1993, 'Induction of nitric oxide synthase in human chodrocytes', Biochemical and Biophysical Research Communication, vol. 193, no. 1, pp. 398-405.
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Incubation of human chondrocytes with interleukin-1?, tumour necrosis factor or endotoxin induced the expression of NO synthase. The synthesis of NO induced by IL-1? was concentration- and time- dependent, occurred after a lag period of approximately 6h and was inhibited by NG-monomethyl-L-arginine, cycloheximide, dexamethasone and hydrocortisone, but not by indomethacin. The activity of NO synthase from activated chondrocytes was not affected by EGTA or by the calmodulin inhibitor W-13. Northern blot analysis, with a rabbit chondrocyte inos probe, showed a 4.4kb positively hybridising band from activated human chondrocytes. Thus, human articular chondrocytes express an inducible NO synthase from the same family as the rabbit chondrocyte and rodent macrophage enzymes. This family appears to vary in terms of in vitro Ca2+-dependence and sensitivity to glucocorticoids.
Cunha, F.Q., Assreuy, J., Xu, D., Charles, I.G., Liew, F.Y. & Moncada, S. 1993, 'Repeated induction of nitric oxide synthase and leishmanicidal activity in murine macrophages', European Journal of Immunology, vol. 23, no. 6, pp. 1385-1388.
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Murine macrophages express high levels of nitric oxide (NO) synthase and produce large amounts of NO when stimulated with interferon-gamma plus lipopolysaccharide in vitro. The expression of NO synthase peaks at 12 h after stimulation and declines rapidly to the background level by 72 h. These macrophages can be repeatedly reactivated to express similar levels of NO synthase. The reactivation is not due to newly divided cells since peritoneal macrophages which do not divide in vitro and J774 cells cultured in the presence of colchicine can also be restimulated to express NO synthase. The reactivation is accompanied by re-expression of NO synthase mRNA, as assessed by polymerase chain reaction analysis. Furthermore, the reactivated macrophages are fully capable of killing the intracellular protozoan parasite Leishmania major.
Anderson, M.D., Fairweather, N., Charles, I.G., Emsley, P., Isaacs, N.W. & McDermott, G. 1993, 'Crystallographic characterization of tetanus toxin fragment C', Journal of Molecular Biology, vol. 230, no. 2, pp. 673-674.
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The C-terminal fragment from tetanus toxin has been crystallized. The 50 kDa protein forms prismatic crystals with an orthorhombic unit cell of dimension a =6403 &Aring;, b=7631 &Aring; and c =1353 &Aring;. The space group is P 212121. Assuming one molecule per asymmetric unit, the solvent occupies 63% of the unit cell.
Brett, S.J., Mazurov, A.V., Charles, I.G. & Tite, J.P. 1993, 'The invasin of Yersinia spp. provides co-stimulatory activity to human T-cells through interaction with beta1- integrins', European Journal of Immunology, vol. 23, no. 7, pp. 1608-1614.
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The invasin proteins of Yersinia spp. are outer membrane proteins which are involved in the penetration of these bacteria into mammalian cells (Cell 1990. 60: 861). Invasin binds to several different beta1 integrins with extremely high affinity, the integrin-binding domain of invasin has been mapped to the C-terminal 192 amino-acids of the molecule (J. Biol. Chem. 1991. 266: 24367). Expression of this fragment alone on the cell surface of non-invasive bacteria is enough to confer the invasive phenotype on these strains (EMBO J. 1990. 9: 1979). Here we show that the carboxy-terminal 192 amino acids of invasin expressed as a fusion protein with the maltose binding protein of E. coli is capable of delivering co-stimulatory signals to human T cells through the beta1 integrins. Co-stimulation was assayed by the ability of invasin to augment the response of highly purified CD4+ and CD8+ T cells to co-immobilized anti-CD3 antibody. Antibody blocking studies indicated that the co-stimulation was mediated through beta1 integrins. The proliferation induced by co-stimulation of CD4+ T cells was accompanied by the synthesis of the cytokines tumor necrosis factor-alpha and interferon-gamma, whereas the activation of CD8+ T cells led to the generation of cytotoxic effectors. The region of the invasin molecule involved in T cell activation was further mapped using synthetic peptides. A region of the invasin molecule containing the residues TAKSKKFPSY could substitute for invasin in T cell activation. The co-stimulation by peptide could also be inhibited by anti-integrin antibodies. The observation that an outer membrane protein of a bacterium which is associated with reactive arthritis and other autoimmune spondyloarthropathies can act as a T cell co-stimulus may have implications for the etiology of these diseases.
Charles, I.G., Chubb, A., Gill, R., Clare, J., Lowe, P., Holmes, L., Page, M., Keeling, J., Moncada, S. & Riverosmoreno, V. 1993, 'Cloning And Expression Of A Rat Neuronal Nitric-Oxide Synthase Coding Sequence In A Baculovirus-Insect Cell System', BIOCHEMICAL AND BIOPHYSICAL RESEARCH C..., vol. 196, no. 3, pp. 1481-1489.
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A DNA sequence encoding rat neurorial NO synthase (nNOS) was isolated and cloned into the baculovirus expression vector pVL1393 to generate pVLRBNOS. Transfection of Spodoptera frugiperda Sf-21 cells with the construct pVLRBNOS resulted in the synthesis of high levels of neuronal NO synthase. Analysis of the expression pattern revealed soluble, enzymatically active NO synthase in the cytoplasm of cell extracts. Active enzyme could also be purified from culture supernatants using 2?-5? ADP sepharose affinity chromatography. This enzyme was recognised by antibodies to the native nNOS and showed a similar degree of inhibition by arginine analogs as the native nNOS. The majority of the NOS synthesised had accumulated as insoluble "inclusion-body" material. The purification of recombinant nNOS from insect cells should facilitate characterisation of neuronal NO synthase
Strugnell, R.A., Dougan, G., Chatfield, S., Charles, I.G., Fairweather, N., Tite, J., Li, J., Beesley, J. & Roberts, M. 1992, 'Characterization Of A Salmonella-Typhimurium-Aro Vaccine Strain Expressing The P.69 Antigen Of Bordetella-Pertussis', Infection And Immunity, vol. 60, no. 10, pp. 3994-4002.
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The P.69 Bordetella pertussis protective antigen was expressed by use of the trc promoter from the chromosome of a Salmonella typhimurium aro vaccine strain, BRD509, by integrating the prn gene, encoding the 93-kDa precursor of this protein, into the aro
Chatfield, S., Fairweather, N., Charles, I.G., Pickard, D., Levine, M., Hone, D., Posada, M., Strugnell, R.A. & Dougan, G. 1992, 'Construction Of A Genetically Defined Salmonella-Typhi Ty2 Aroa, Aroc Mutant For The Engineering Of A Candidate Oral Typhoid Tetanus Vaccine', Vaccine, vol. 10, no. 1, pp. 53-60.
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The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no
Li, J., Fairweather, N., Novotny, P., Dougan, G. & Charles, I.G. 1992, 'Cloning, Nucleotide-Sequence And Heterologous Expression Of The Protective Outer-Membrane Protein P.68 Pertactin From Bordetella-Bronchiseptica', Journal Of General Microbiology, vol. 138, no. NA, pp. 1697-1705.
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The prn gene encoding the 68 kDa protective outer-membrane protein of Bordetella bronchiseptica (P.68 pertactin) was cloned, sequenced and expressed in Escherichia coli. The gene was isolated by DNA: DNA hybridization experiments using a radioactively-la
Chatfield, S., Charles, I.G., Makoff, A., Oxer, M., Dougan, G., Pickard, D., Slater, D. & Fairweather, N. 1992, 'Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains - Development of a single-dose oral tetanus vaccine', Nature Biotechnology, vol. 10, no. 8, pp. 888-892.
Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509. The resulti
Roberts, M., Tite, J., Fairweather, N., Dougan, G. & Charles, I.G. 1992, 'Recombinant P69/Pertactin - Immunogenicity And Protection Of Mice Against Bordetella-Pertussis Infection', Vaccine, vol. 10, no. 1, pp. 43-52.
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The immunogenicity of recombinant (r-) pertactin was examined. Parenteral immunization of mice with natural or r-pertactin produced a similar increase in serum anti-pertactin antibodies and a decrease in Bordetella pertussis lung counts following aeroso
Vandenhombergh, J., Moore, J., Charles, I.G. & Hawkins, A. 1992, 'Overproduction In Escherichia-Coli Of The Dehydroquinate Synthase Domain Of The Aspergillus-Nidulans Pentafunctional Arom Protein', Biochemical Journal, vol. 284, no. NA, pp. 861-867.
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The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3
Chatfield, S., Strahan, K., Pickard, D., Charles, I.G., Hormaeche, C. & Dougan, G. 1992, 'Evaluation Of Salmonella-Typhimurium Strains Harboring Defined Mutations In Htra And Aroa In The Murine Salmonellosis Model', Microbial Pathogenesis, vol. 12, no. 2, pp. 145-151.
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Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harbouring defined mutations in htrA, aroA or htrA aroA combined. When administered orally or intravenously to BALB/c mice, all the mutants were found to be highly attenuated. All mutants were able to confer significant protection against lethal challenge with SL1344 after a single oral dose of live organisms. SL1344 htrA mutants persisted in livers and spleens at a lower level than SL1344 aroA mutants after intravenous administration. SL1344 htrA aroA mutants persisted at an even lower level and were cleared from the livers and spleens of mice within 21 days of intravenous administration. Thus htrA and htrA aroA mutants can be considered as potential oral vaccines against salmonellosis.
Boys, C.W., Bury, S.M., Sawyer, L., Moore, J.D., Charles, I.G., Hawkins, A.R., Deka, R., Kleanthous, C. & Coggins, J.R. 1992, 'Crystallization of a type I 3-dehydroquinase from Salmonella typhi', Journal of Molecular Biology, vol. 227, no. 1, pp. 352-355.
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Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 23 &Aring; on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P21212 with unit cell dimensions a = 4801 &Aring;, B = 11429 &Aring;, C = 4287 &Aring;. There is one subunit in the asymmetric unit.
Peakman, T.C., Charles, I.G., Sydenham, M.A., Gewert, D.R., Page, M.J. & Makoff, A.J. 1992, 'Enhanced expression of recombinant proteins in insect cells using a baculovirus vector containing a bacterial leader sequence', Nucleic Acids Research, vol. 20, no. 22, pp. 6111-6112.
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Baculovirus/insect cell expression systems have been in use for several years, often resulting in very high levels of_expression of recombinant proteins (1). For expression of mature proteins the preferred vectors, such as pVL941 (2) or p36C (3), utilise the strong polyhedrin promoter and leader sequence. We recently reported the finding that the presence of a 24 bp bacterial sequence immediately upstream of the initiation codon unexpectedly supported high expression levels of tetanus toxin fragment C (4). The bacterial leader sequence is derived from the 3' end of the E.coli trpE gene where it functions as part of the ribosome binding site of the adjacent trpD gene (5). This sequence also enabled P69 pertactin from Bordetella pertussis to be expressed -at high levels by baculovirus~infected cells (unpublished observations). We have investigated this bacterial sequence further. by comparing its effect on the expression of three different heterologous genes.
Moore, J.D., Lamb, H.K., Garbe, T., Servos, S., Dougan, G., Charles, I.G. & Hawkins, A.R. 1992, 'Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis', Biochemical Journal, vol. 287, no. 1, pp. 173-181.
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The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable, is active as a homododecamer and has the lowest reported Km value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA- qutB- double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE- mutant strain of A. nidulans to utilize quinate as sole carbon source.
Garbe, T., Servos, S., Hawkins, A., Dimitriadis, G., Young, D., Dougan, G. & Charles, I.G. 1991, 'The Mycobacterium-Tuberculosis Shikimate Pathway Genes - Evolutionary Relationship Between Biosynthetic And Catabolic 3-Dehydroquinases', Molecular & General Genetics, vol. 228, no. 3, pp. 385-392.
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The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate
Novotny, P., Chubb, A., Cownley, K. & Charles, I.G. 1991, 'Biologic and protective properties of the 69-kda outer-membrane protein of Bordetella-Pertussis - A novel formulation for an acellular pertussis-vaccine', Journal of Infectious Diseases, vol. 164, no. 1, pp. 114-122.
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A combination of the 69-kDa outer membrane protein and filamentous hemagglutinin (FHA), both isolated from lymphocytosis promoting factor (LPF; pertussis toxin) minus mutants of Bordetella pertussis, is protective in the mouse intracerebral challenge pot
Charles, I.G., Rodgers, B., Makoff, A., Chatfield, S., Slater, D. & Fairweather, N. 1991, 'Synthesis Of Tetanus Toxin Fragment C In Insect Cells By Use Of A Baculovirus Expression System', Infection And Immunity, vol. 59, no. 5, pp. 1627-1632.
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The baculovirus expression vector p36C was used to express in cells of the insect Spodoptera frugiperda fragment C of tetanus toxin under the control of the strong polyhedrin promoter. Fragment C was expressed intracellularly at a high level and was sol
Leininger, E., Roberts, M., Kenimer, J., Charles, I.G., Fairweather, N., Novotny, P. & Brennan, M. 1991, 'Pertactin, An Arg-Gly-Asp-Containing Bordetella-Pertussis Surface Protein That Promotes Adherence Of Mammalian-Cells', Proceedings Of The National Academy Of Sciences ..., vol. 88, no. 2, pp. 345-349.
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A 69-kDa protein has been identified on the surface of the Gram-negative pathrogen Bordetella pertussis that can elicit a protective immune response in animal models. This protein is associated with virulent strains of B. pertussis but its function has remained unclear. In this report we demonstrate that purified preparations of the 69-kDa outer membrane protein can promote the attachment of Chinese hamster ovary (CHO) cells. The interaction between the mammalian cells and this protein can be specifically inhibited by an Arg-Gly-Asp (RGD)-containing synthetic peptide that is homologous with a region found in the 69-kDa protein sequence. These studies indicate that a specific cell binding site containing an Arg-Gly-Asp sequence may be involved in the interaction of this bacterial protein with mammalian cell surfaces. To further investigate the role of this protein as a bacterial adhesin, a mutant of B. pertussis W28 that does not express the 69-kDa protein was constructed using the plasmid vector pRTP1. This mutant was 30-40% less efficient at adhering to CHO cells and to human HeLa cells than was the parent strain. These data support a role for this 69-kDa outer membrane protein in the attachment of B. pertussis to mammalian cells. We propose the name "pertactin" for this protein.
Oxer, M.D., Bentley, C.M., Doyle, J.G., Peakman, T.C., Charles, I.G. & Makoff, A.J. 1991, 'High level heterologous expression in E.coli using the anaerobically-activated nirB promoter', Nucleic Acids Research, vol. 19, no. 11, pp. 2889-2892.
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The anaerobically-regulated nirB promoter was used to express heterologous genes in Escherichia coli. Under anaerobic conditions the promoter was able to express tetanus toxin fragment C at approximately 20% total cell protein (tcp) and the Bordetella pertussis antigen pertactln at greater than 30% tcp. These levels are comparable to those obtained for the same products using the tac promoter. The nirB promoter Is very well regulated, giving almost two orders Of magnitude Increase in fragment C on complete removal of oxygen. The use of this anaerobically-Induced promoter in the production of recombinant proteins In E. coli is discussed.
Servos, S., Chatfield, S., Hone, D., Levine, M., Dimitriadis, G., Pickard, D., Dougan, G., Fairweather, N. & Charles, I.G. 1991, 'Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi', Journal Of General Microbiology, vol. 137, no. 1, pp. 147-152.
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The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated Mr of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-reacted with its S. typhi counterpart.
Servos, S., Silva, C., Dougan, G. & Charles, I.G. 1991, 'The commercially available restriction enzyme BspHl is blocked by overlapping methylation', Nucleic Acids Research, vol. 19, no. 1, pp. 183-183.
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The restriction endonuclease Bspill. conunercially available from New England Biolabs (Bishop's Stortford, UK), cleaves the site T/CATGA. This restriction enzyme is of particular use in the construction of gene expression cassettes in the vector pKK233 -2 (I) as it is one of the three enzymes along with AflIII and NcoI that has an 'ATG' in its recognition sequence. In this paper we show that BspHI is Dam methylase (2) sensitive and will not cleave DNA that has the overlapping sequ~nce GATC. The oligonucleotides: OS3142 5' -CTAGATCATGAGCGAACTG~ TCGTGAACGTGATCAAAGCTTGGTAC- 3' and OS3143 5'-CAAGCTTTGATCACGTTCACGATCAGTTCGCTCATGAT- 3' were annealed and ligated into XbaI-KpnI digested bluescript SKI1+ (Stratagene, Cambridge, UK}.
Romanos, M., Clare, J., Beesley, K., Rayment, F., Ballantine, S., Makoff, A., Dougan, G., Fairweather, N. & Charles, I.G. 1991, 'Recombinant Bordetella-Pertussis Pertactin (P69) From The Yeast Pichia-Pastoris - High-Level Production And Immunological Properties', Vaccine, vol. 9, no. 12, pp. 901-906.
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Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to hig
Novotny, P., Chubb, A.P., Cownley, K. & Charles, I.G. 1991, 'A novel bivalent acellular pertussis vaccine based on the 69 kDa protein and FHA', Developments in biological standardization, vol. 73, pp. 243-249.
Charles, I.G., Li, J.L., Dougan, G., Pickard, D., Francis, M., Romanos, M., Beesley, K., Brennan, M., Manclark, M.R., Jensen, M., Heron, I., Chubb, A., Novotny, P. & Fairweather, N. 1991, 'Identification and characterization of a protective immunodominant B cell epitope of pertactin (P.69) from Bordetella pertussis', European Journal of Immunology, vol. 21, pp. 1147-1153.
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Epitopes defined by monoclonal antibodies (mAb) specific for the Bordetella pertussis outer membrane protein P.69 (pertactin) were mapped using a series of amino- and carboxy-terminal deletion mutants expressed in Escherichia coli. mAb were found to bind predominantly to a region of pertactin spanning a (Pro-Gln-Pro)5 repeat motif and one mAb was found to bind to another region spanning a (Gly-Gly-X(aa)-X(aa)-Pro)5 repeat motif. To localize further the mAb-binding sites a panel of synthetic peptides, a series of 94 overlapping hexameric peptides, and a P.69 30-amino acid fusion to a hepatitis B core protein (HBcAg-69), were synthesized. This combined approach has identified the binding site for the mAb BBO5: Pro-Gly-Pro-Gln-Pro-Pro; mAb BBO7, E4A8 and E4D7: Ala-Pro-Gln-Pro-Pro-Ala-Gly-Arg; and mAb BPE3: Thr-Leu-Trp-Tyr-Ala-Glu-Ser-Asn-Ala-Leu-Ser-Lys-Arg. We have used a non-lethal murine respiratory model of B. pertussis infection to investigate the ability of a peptide containing the epitope of the mAb BBO5 to elicit protective immunity. Immunization of mice with the HBcAg-69 protein prevented growth of B. pertussis in the lungs compared to mice receiving HBcAg alone, and protection correlated with high titers of anti-P.69 antibodies.
Roberts, M., Fairweather, N., Leininger, E., Pickard, D., Hewlett, E., Robinson, A., Hayward, C., Dougan, G. & Charles, I.G. 1991, 'Construction and characterisation of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane protein', Molecular Microbiology, vol. 5, no. 6, pp. 1393-1404.
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The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69. The cloned P.69 (prn) gene of B. pertussis was insertionally inactivated with a kanamycin-resistance cassette. This inactivated gene was used to construct P.69- mutants of B. pertussis by allelic exchange using plasmid pRTP1. B. pertussis P.69- strains produced normal levles of other vir-regulated factors, including adenylate cyclase. The serotype of B. pertussis, determined by Eldering and Preston typing sera and monoclonal antibodies, was also unaffected by the presence or absence of P.69. The ability of a prn mutant to adhere to and invade HEp2 cells was not significantly different from that of its parent strain. A strain containing a mutation in fhaB was significantly less adhesive and invasive than its parent, and a prn fhaB double mutant exhibited an even greater reduction in adhesiveness and invasiveness down to levels comparable with a Vir- strain. However, strains harbouring mutations in FHA and/or P.69 were able to colonize or multiply in the murine respiratory tract, although a Vir- strain was unable to survive and proliferate in the same infection model.
Coleman, D., Knights, J., Pickard, D., Carroll, D., Birkbeck, T.M., Dougan, G. & Charles, I.G. 1991, 'Insertional inactivation of the Staphylococcus aureusβ-toxin by bacteriophage o13 occurs by site-and orientation-specific integration of the o13 genome', Molecular Microbiology, vol. 5, no. 4, pp. 933-939.
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Lysogenization of Staphylococcus aureus by the sero-type F converting bacteriophage phi-13 results in loss of beta-toxin expression. Sequence analysis of the S. aureus beta-toxin gene (hlb), the attachment site (attP)-containing region of phi-13 DNA and the chromosome/bacteriophage DNA junctions of a phi-13 lysogen, revealed that the molecular mechanism of loss of beta-toxin expression was due to insertion of the phi-13 genome into the 5' end of hlb. The insertion site (attB) within hlb contained a 14 base pair core sequence in common with attP and both ends of the integrated linear prophage genome of a phi-13 lysogen. These findings indicate that integration of the phi-13 genome into hlb is site- and orientation-specific
Johnson, K., Charles, I.G., Dougan, G., Pickard, D., O'Goara, P., Costa, G., Ali, T., Miller, I. & Hormaeche, C.E. 1991, 'The role of stress response protein in Salmonella typhimurium virulence', Molecular Microbiology, vol. 5, no. 2, pp. 401-407.
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We recently described the use of selective transposon mutagenesis to generate a series of avirulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat-shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated M(r) of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature-sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.
Lipscombe, M., Charles, I.G., Roberts, M., Dougan, G., Tite, J. & Fairweather, N. 1991, 'Instranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen', Molecular Microbiology, vol. 5, pp. 1385-1392.
The plasmid pBRD026, which directs expression of the B subunit of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3' end of the gene. An oligonucleotide linker containing restriction sites for BglII and SpeI was inserted at the SpeI site at the 3' end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly-Pro-Gly-Pro which we propose acts as a 'hinge' between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetella pertussis P.96 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.
Garbe, T., Jones, C., Charles, I.G., Dougan, G. & Young, D. 1990, 'Cloning and Characterization of the aroA Gene from Mycobacterium tuberculosis', Journal Of Bacteriology, vol. 172, no. 12, pp. 6774-6782.
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The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines.
Charles, I.G. & Dougan, G. 1990, 'Gene expression and the development of live enteric vaccines', Trends In Biotechnology, vol. 8, pp. 117-121.
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Rationally attenuated liveSalmonella vaccines provide good protection against homologous challenge and can act as carriers of heterologous antigens. However, the optimum expression system for each heterologous antigen will need to be established individually. This will ensure that the antigen in question is produced at appropriate levels, in the correctly folded conformation, within or at the surface of the carrier cell, and can therefore elicit the optimal immune response.
Fairweather, N., Chatfield, S., Charles, I.G., Roberts, M., Lipscombe, M., Li, L., Strugnell, D., Comerford, S., Tite, J. & Dougan, G. 1990, 'Use Of Live Attenuated Bacteria To Stimulate Immunity', Research in Microbiology, vol. 141, no. 7-8, pp. 769-773.
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Many bacteria, including Salmonella, Listeria, and Yersinia species, can cause severe systemic infections of mammals. These invasive bacteria appear to have developed sophisticated mechanisms for entry into, and survival within, eukaryotic cells. Genetic manipulation of these organisms has led to the isolation of mutants which have an impaired ability to survive in vivo, and some of these mutants have been used as live oral vaccines to immunize against virulent infection. In this review, we shall outline some of the work carried out in our laboratory on the analysis of these bacteria, including the construction of auxotrophic mutants and their use in carrying foreign antigens to the immune system. In addition, the use of secreted proteins as immunogens and as carriers of epitopes will be discussed.
Makoff, A., Oxer, M., Ballantine, S., Fairweather, N. & Charles, I.G. 1990, 'Protective surface antigen-P69 of Bordetella pertussis - Its characterization and very high-level expression in Escherichia coli', Nature Biotechnology, vol. 8, no. 11, pp. 1030-1033.
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The surface antigen, P69 of Bordetella pertussis, an N-terminal fragment of the precursor protein, P93, is likely to be an important component of future subunit vaccines against whooping cough. We have expressed several defined N-terminal fragments of P93 in E. coli and compared their electrophoretic mobilities with that of purified P69 from B. pertussis. These experiments show that P69 is considerably smaller than the 69 kD originally estimated from its gel mobility and is probably 60.4 kD in size. Our initial plasmids expressed only very low levels of this antigen. We diagnosed the limiting factor to be a poor ribosome binding site (RBS) by demonstrating a large stimulation of expression on a two-cistron plasmid. The limitation of expression could be completely overcome by only two base changes close to the initiation codon, resulting in a further increase in expression of P69 at levels to 30&frac12;40% total cell protein. Although the protein accumulated as insoluble inclusion bodies, it could be solubilized by guanidinium chloride.
Charles, I.G., Lamb, H., Pickard, D., Dougan, G. & Hawkins, A. 1990, 'Isolation, Characterization And Nucleotide-Sequences Of The Aroc Genes Encoding Chorismate Synthase From Salmonella-Typhi And Escherichia-Coli', Journal Of General Microbiology, vol. 136, no. NA, pp. 353-358.
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The aroC genes from Salmoneffa @phi and Escherichia cofi, encoding 5-enolpyruvylshikimate-3-phosphate phospholyase (chorismate synthase) were cloned in E. coli and their DNA sequences were determined. The aroC gene from S @phi was isolated from a cosmid gene bank by complementation of an E. cofi aroC mutant. The corresponding E. coli gene was isolated from a pBR322 gene bank by colony hybridization using DNA encoding the aroC gene from S. @phi as a hybridization probe. Analysis of the nucleotide sequence revealed that both genes have an open reading frame capable of encoding proteins comprising 361 amino acids. The calculated molecular mass of the protein from S fyphi is 39 10s Da while that of the protein from E cofi is 39 138 Da. Homology is particularly strong between the coding regions of the genes: 95% when protein sequences are compared, and 83% when DNA sequences are examined. Use of a deletion variant of the E. cofi aroC gene demonstrates that the C-terminal36 amino acids are not essential for the correct folding or functional activity of the chorismate synthase enzyme.
Chatfield, S., Dougan, G. & Charles, I.G. 1990, 'Complete nucleotide sequence of the aroA gene from Salmonella typhi encoding 5-enolpyruvyishikimate 3-phosphate synthase', Nucleic Acids Research, vol. 18, no. 20, pp. 6133-6133.
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As part of a programme of research to generate fully characterized aro- mutants of Salmonella typhi for use as live attenuated vaccines we have cloned and sequenced the aroC (1) and aroD genes (submitted to J. Gen. Microbiol.) of S. typhi. We report here the nucleotide sequence of the S. typhi aroA gene encoding 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase).
Dougan, G., Chatfield, S.N., Roberts, M., Charles, I.G., Comerford, S., Li, L.J. & Fairweather, N. 1990, 'Bacterial pathogens: A route to oral drug delivery', Biochemical Society Transactions, vol. 18, pp. 746-748.
Li, L.J., Dougan, G., Novotny, P. & Charles, I.G. 1990, 'Polymerease chain reaction (PCR) allows the specific detection of a single cell of Bordetella pertussis', Life Sciences Advances, vol. 9, pp. 41-46.
Johnson, K., Charles, I.G., Dougan, G., Miller, I., Pickard, D., Ogoara, P., Costa, G., Ali, T. & Hormaeche, C. 1990, 'The Role Of A Stress-Response Protein In Bacterial Virulence', Research in Microbiology, vol. 141, no. 7-8, pp. 823-825.
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We have utilised the transposon TnphoA to generate Salmonella typhimurium mutants of reduced virulence when given orally to mice (Miller et ai., 1989). S. typhimurium 014 and 046 are two such attenuated derivatiees of the mouse-virulent strain C5, being approximately 3-6 orders of magnitude less virulent than the parent. They were originally thought to have different properties (Miller et al., 1989), but subsequent analysis revealed them to the identical (our unpublished observations). Both mutants were insensitive to the cytolytic effects of serum plus complement and were unaltered in ,~their ability to invade. MDCK cell monolayers (B. Fmlay," pets. comm.). In vivo, ,ae mutants localized in the livers and spleens of infected mice, whether administered by intravenous or oral routes, though the number of bacteria that could be recow~red from infected tissues was ca. 105-fold lower than observed with the parent strain. In this work we further characterize the lesion in these mutants.
Charles, I.G., Dougan, G., Pickard, D., Chatfield, S., Smith, M.A., Novotny, P., Morrissey, P.E. & Fairweather, N. 1989, 'Molecular-Cloning And Characterization Of Protective Outer-Membrane Protein-P.69 From Bordetella-Pertussis', Proceedings Of The National Academy Of Sciences Of The United States Of America, vol. 86, no. 10, pp. 3554-3558.
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Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.
Novotny, P., Chubb, A.P., Cownley, K., Charles, I.G., Heart, T.A., Macaulay, M. & Skaril, F. 1989, 'Prospects for a 69kDa protein based subcellular pertussis vaccine', Journal of Medical Microbiology, vol. 35, pp. 186-186.
Dougan, G., Maskell, D., Ocallaghan, D., Chatfield, S., Charles, I.G. & Hormaeche, C. 1988, 'Oral Vaccination', Antonie Van Leeuwenhoek Journal Of Microbiology, vol. 54, no. 5, pp. 447-451.
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At present most generally available bacterial vaccines are composed of whole killed microorganisms which are administered parenterally with or without simple adjuvants (1). Such vaccines can be highly efficaceous, for example, the current whooping cough and tetanus vaccines, but on the whole these crude vaccines have either poor efficacy or induce side effects in vaccinees. The majority of bacterial pathogens infect the host either at or via mucosal surfaces, and in vaccinating against them induction of an immune response at this site is of great importance. However, parenteral immunization is a poor route to choose to induce mucosal immunity (2). This may explain in part the low effectiveness of parenteral immunization against such pathogens as Vibrio cholerae (3). Parenteral administration of crude vaccines containing potentially toxic components such as lipopolysaccharide often results in side-effects ranging from inflammation at the site of injection to severe, systemic morbidity. An alternative to parenteral administration is to deliver antigens via the oral route. Oral vaccination, especially with live attenuated organisms, can stimulate the gut associated lymphoid tissue resulting in the induction of mucosal immunity which is particularly important in protection against enteric pathogens. Oral vaccination also overcomes side-effects caused by parenteral injection of vaccines. In this paper we will discuss the potential of oral vaccination against enteric pathogens.
Charles, I.G., Li, L.J., Dougan, G., Pickard, D., Chatfield, S.N., Smith, M., Novotny, P. & Fairweather, N. 1988, 'Molecular cloning and analysis of P.69, a vir controlled protein from Bordetella pertussis', Tokai Journal of Experimental and Clinical Medicine, vol. 13, pp. 227-234.
Creighton, T.E. & Charles, I.G. 1987, 'Biosynthesis, Processing, And Evolution Of Bovine Pancreatic Trypsin-Inhibitor', Cold Spring Harbor Symposia On Quantitative Biology, vol. 52, no. NA, pp. 511-519.
Functional products of gene expression are generally obtained only after the initial linear polypeptide chain has folded to its native three-dimensional conformation (Creighton 1984). Unfolded proteins are usually inactive biologically. Folding is believed to be a spontaneous self-assembly process, directed solely by the amino acid sequence of the polypeptide chain under the appropriate physiological conditions, and occurs shortly after assembly of the polypeptide chain on the ribosome.
Brammar, W.J., Charles, I.G., Matfield, M., Cheng-Pin, L., Drew, R. & Clarke, P.H. 1987, 'The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa', FEBS Letters, vol. 215, no. 2, pp. 291-294.
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The nucleotide sequence of the amiE gene, encoding the aliphatic amidase of Pseudomonas aeruginosa, has been determined. The sequence of 1038 nucleotides shows a strong bias in favour of codons with G or C in the third position, and only 44 different codons are utilised
Creighton, T.E. & Charles, I.G. 1987, 'Sequences Of The Genes And Polypeptide Precursors For 2 Bovine Protease Inhibitors', Journal of Molecular Biology, vol. 194, no. 1, pp. 11-22.
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The genes for bovine pancreatic trypsin inhibitor and a homologous protease inhibitor have been characterized using messenger RNA, clones of complementary DNA copies and genomic fragments. Both genes consist of three exons and two introns and are virtually identical in sequence, suggesting that recently they either arose by gene duplication or were subject to gene conversion. However, they differ markedly in their promoter regions and in a segment within the first intron. Perhaps as a consequence, the two genes can be expressed using different transcription start sites, and the two proteins are found in different bovine tissues. Each middle exon encodes primarily the mature protein, while the other two exons define amino- and carboxyl-terminal extensions of 33 or 35 and 7 amino acid residues, respectively. The amino-terminal extensions contain a signal peptide-like sequence, suggesting that the proteins are destined for cellular compartments. The remaining extensions at both ends may be involved in targetting of the proteins, and there are some similarities to other proteins destined for cellular compartments.
Boyd, A.J., Charles, I.G., Keyte, J. & Brammar, W. 1986, 'Isolation And Computer-Aided Characterization Of Mmei, A Type-Ii Restriction Endonuclease From Methylophilus-Methylotrophus', Nucleic Acids Research, vol. 14, no. 13, pp. 5255-5274.
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A Type II restriction endonuclease, MmeI, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-palindromic recognition sequence 5'-T C C Pu A C (N)20-3', 3'-A G G Py T G (N)18-5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved using two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules.
Charles, I.G., Keyte, J.W., Brammar, W.J., Smith, M. & Hawkins, A.R. 1986, 'The isolation and nucleotide sequence of the complex AROM locus of Aspergilus nidulans', Nucleic Acids Research, vol. 14, no. 5, pp. 2201-2213.
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The AROM locus of A.nidulans, which governs five consecutive steps in pre-chorismate aromatic amino acid biosynthesis, has been cloned in a bacteriophage vector. The nucleotide sequence of the locus reveals a single, open reading-frame of 4,812 base-pairs, apparently without introns. An internal segment of the A.nidulans AROM sequence has extensive homology with the E.coli aroA gene that encodes the 5-enolpyruvylshikimate 3-phosphate synthase.
Charles, I.G., Harford, S., Brookfield, J. & Shaw, W. 1985, 'Resistance To Chloramphenicol In Proteus-Mirabilis By Expression Of A Chromosomal Gene For Chloramphenicol Acetyltransferase', Journal Of Bacteriology, vol. 164, no. 1, pp. 114-122.
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Proteus mirabilis PM13 is a well-characterized chloramphenicol-sensitive isolate which spontaneously gives rise to resistant colonies on solid media containing chloramphenicol (50 micrograms ml-1) at a plating efficiency of 10(-4) to 10(-5). Such chloramphenicol-resistant colonies exhibit a novel phenotype with respect to chloramphenicol resistance. When a single colony grown on chloramphenicol agar is transferred to liquid medium and grown in the absence of antibiotic for 150 generations, a population of predominantly sensitive cells arises. This mutation-reversion phenomenon has been observed in other Proteus species and Providencia strains, wherein resistance has been shown to be mediated in each case by the enzyme chloramphenicol acetyltransferase. The cat gene responsible for the phenomenon is chromosomal and can be cloned from P. mirabilis PM13 with DNA prepared from cells grown in the absence or the presence of chloramphenicol. Recombinant plasmids which confer resistance to chloramphenicol carry an 8.5-kilobase PstI fragment irrespective of the source of host DNA. The location of the cat gene within the PstI fragment was determined by Southern blotting with a cat consensus oligonucleotide corresponding to the expected amino acid sequence of the active site region of chloramphenicol acetyltransferase, and the direction of transcription was deduced from homology with the type I cat variant.
Charles, I.G., Keyte, J. & Shaw, W. 1985, 'Nucleotide-Sequence Analysis Of The Cat Gene Of Proteus-Mirabilis - Comparison With The Type-I (Tn9) Cat Gene', Journal Of Bacteriology, vol. 164, no. 1, pp. 123-129.
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In Proteus mirabilis PM13 chloramphenicol resistance is mediated by the cat gene, a single copy of which is present in both resistant and sensitive isolates and which reverts at a high frequency. RNA measurements show an about 8.5-fold increase in cat-specific mRNA in cells expressing the resistance phenotype as compared with those which are sensitive to chloramphenicol. DNA sequence analysis has revealed a high degree of homology between the P. mirabilis cat gene and the type I cat variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. Sequence homology between the strain PM13 cat variant and Tn9 cat was not apparent however in the 5' and 3' flanking regions. Segments of near identity were seen when the upstream sequence of the cat of P. mirabilis was compared with the 5' regions of the Salmonella typhimurium flagellin genes HI and H2, which are alternately expressed by a flip-flop control mechanism involving an invertible promoter and a trans-acting product.
Charles, I.G., Keyte, J., Brammar, W. & Hawkins, A. 1985, 'Nucleotide-Sequence Encoding The Biosynthetic Dehydroquinase Function Of The Penta-Functional Arom Locus Of Aspergillus-Nidulans', Nucleic Acids Research, vol. 13, no. 22, pp. 8119-8128.
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The nucleotide sequence of a 1.9 Kb HindIII fragment of DNA derived from the AROM locus of A.nidulans and encoding the biosynthetic dehydroquinase activity has been determined. The sequences encoding the biosynthetic and catabolic dehydroquinase enzymes of A.nidulans show no detectable homology, strongly suggesting convergent evolutionary pathways. The messenger RNA specified by the AROM locus was detected a3 a 5.3 Kb RNA species.
Beschle, H.G., Charles, I.G. & Shaw, W.V. 1984, 'Use of a Synthetic Oligonucleotide to Identify a Chromosomal Gene for Chloramphenicol Acetyltransferase in a Plasmid-bearing Flavobacterium', Journal of General Microbiology, vol. 130, no. NA, pp. 3335-3338.
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A micro-organism previously designated Flavobacterium sp. CB60 is resistant to chloramphenicol as a consequence of antibiotic acetylation by the enzyme chloramphenicol acetyltransferase and subsequent degradation of the acetylated product by co-metabolism. Although a 15.6 kb plasmid (pCB60) was demonstrated in this Flavobacterium strain, it did not appear to play a role in chloramphenicol acetylation. DNA hybridization was used to identify a fragment of DNA presumptively carrying the cat gene. The sequence of the synthetic probe was based upon known nucleotide sequences corresponding to the highly-conserved active site region of several chloramphenicol acetyltransferase variants. The structural gene for the enzyme in strain CB60 appeared to be chromosomal since the radioactive probe hybridized with a unique restriction fragment from chromosomal DNA but failed to do so with the DNA of plasmid pCB60.