428 results on '"Richard W. Titball"'
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2. The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin
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Richard W. Titball
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Clostridium perfringens ,ε-toxin ,pore-forming toxin ,structure ,receptor ,Medicine - Abstract
Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.
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- 2024
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3. Strep A: challenges, opportunities, vaccine-based solutions and economics
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David E. Bloom, Richard W. Titball, and Jonathan Carapetis
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Immunologic diseases. Allergy ,RC581-607 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Published
- 2024
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4. Coxiella burnetii replicates in Galleria mellonella hemocytes and transcriptome mapping reveals in vivo regulated genes
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Andrea Kovacs-Simon, Georgie Metters, Isobel Norville, Claudia Hemsley, and Richard W. Titball
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coxiella burnetii ,galleria mellonella ,t4ss ,virulence ,transcriptome profiling ,Infectious and parasitic diseases ,RC109-216 - Abstract
Larvae of the greater wax moth (Galleria mellonella) are susceptible to infection with C. burnetii, an obligate intracellular bacterial pathogen. We show that bacteria are found in hemocytes after infection, and occupy vacuoles which are morphologically similar to Coxiella-containing vacuoles seen in infected mammalian phagocytes. We characterized the infection by transcriptome profiling of bacteria isolated from the hemocytes of infected larvae and identified 46 highly upregulated genes. The encoded proteins are predicted to be involved in translation, LPS biosynthesis, biotin synthesis, scavenging of reactive oxygen species, and included a T4SS effector and 30 hypothetical proteins. Some of these genes had previously been shown to be upregulated in buffalo green monkey (BGM) cells or in mice, whilst others appear to be regulated in a host-specific manner. Altogether, our results demonstrate the value of the G. mellonella model to study intracellular growth and identify potential virulence factors of C. burnetii.
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- 2020
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5. The rapid progress in COVID vaccine development and implementation
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Alan D. T. Barrett, Richard W. Titball, Paul A. MacAry, Richard E. Rupp, Veronika von Messling, David H. Walker, and Nicolas V. J. Fanget
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Immunologic diseases. Allergy ,RC581-607 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Published
- 2022
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6. The pore structure of Clostridium perfringens epsilon toxin
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Christos G. Savva, Alice R. Clark, Claire E. Naylor, Michel R. Popoff, David S. Moss, Ajit K. Basak, Richard W. Titball, and Monika Bokori-Brown
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Science - Abstract
Epsilon toxin (Etx) is a potent pore forming toxin (PFT) produced by Clostridium perfringens. Here authors show the cryo-EM structure of the Etx pore assembled on the membrane of susceptible cells and shed light on pore formation and mutant phenotypes.
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- 2019
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7. Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups
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Claudia M. Hemsley, Paul A. O’Neill, Angela Essex-Lopresti, Isobel H. Norville, Tim P. Atkins, and Richard W. Titball
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Coxiella burnetii ,Whole Genome Sequencing ,Genotyping ,Pan-Genome Analysis ,Patho-adaptation ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. Results Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. Conclusions The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.
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- 2019
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8. Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model
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Drew Adler, Jennifer R. Linden, Samantha V. Shetty, Yinghua Ma, Monika Bokori-Brown, Richard W. Titball, and Timothy Vartanian
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Science - Abstract
Summary: Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor. : Pathogenic Organism; Vascular Remodeling; Molecular Mechanism of Behavior; Model Organism Subject Areas: Pathogenic Organism, Vascular Remodeling, Molecular Mechanism of Behavior, Model Organism
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- 2019
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9. Galleria mellonella as an infection model to investigate virulence of Vibrio parahaemolyticus
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Sariqa Wagley, Richard Borne, Jamie Harrison, Craig Baker-Austin, Donatella Ottaviani, Francesca Leoni, Varaporn Vuddhakul, and Richard W. Titball
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Vibrio parahaemolyticus ,nudix hydrolase ,MutT ,Galleria mellonella ,Infectious and parasitic diseases ,RC109-216 - Abstract
Non-toxigenic V. parahaemolyticus isolates (tdh−/trh−/T3SS2−) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mellonella) are susceptible to infection by toxigenic or non-toxigenic clinical isolates of V. parahaemolyticus. In comparison larvae inoculated with environmental isolates of V. parahaemolyticus did not succumb to disease. Whole genome sequencing of clinical non-toxigenic isolates revealed the presence of a gene encoding a nudix hydrolase, identified as mutT. A V. parahaemolyticus mutT mutant was unable to kill G. mellonella at 24 h post inoculation, indicating a role of this gene in virulence. Our findings show that G. mellonella is a valuable model for investigating screening of possible virulence genes of V. parahaemolyticus and can provide new insights into mechanisms of virulence of atypical non-toxigenic V. parahaemolyticus. These findings will allow improved genetic tests for the identification of pathogenic V. parahaemolyticus to be developed and will have a significant impact for the scientific community.
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- 2018
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10. Isolation and primary culture of Galleria mellonella hemocytes for infection studies [version 2; peer review: 2 approved]
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Nicola J. Senior and Richard W. Titball
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Medicine ,Science - Abstract
Galleria mellonella larvae are increasingly used to study the mechanisms of virulence of microbial pathogens and to assess the efficacy of antimicrobials. The G. mellonella model can faithfully reproduce many aspects of microbial disease which are seen in mammals, and therefore allows a reduction in the use of mammals. The model is now being widely used by researchers in universities, research institutes and industry. An attraction of the model is the interaction between pathogen and host. Hemocytes are specialised phagocytic cells which resemble neutrophils in mammals and play a major role in the response of the larvae to infection. However, the detailed interactions of hemocytes with pathogens is poorly understood, and is complicated by the presence of different sub-populations of cells. We report here a method for the isolation of hemocytes from Galleria mellonella. A needle-stick injury of larvae, before harvesting, markedly increased the recovery of hemocytes in the hemolymph. The majority of the hemocytes recovered were granulocyte-like cells. The hemocytes survived for at least 7 days in culture at either 28°C or 37°C. Pre-treatment of larvae with antibiotics did not enhance the survival of the cultured hemocytes. Our studies highlight the importance of including sham injected, rather than un-injected, controls when the G. mellonella model is used to test antimicrobial compounds. Our method will now allow investigations of the interactions of microbial pathogens with insect hemocytes enhancing the value of G. mellonella as an alternative model to replace the use of mammals, and for studies on hemocyte biology.
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- 2021
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11. Isolation and primary culture of Galleria mellonella hemocytes for infection studies [version 1; peer review: 2 approved]
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Nicola J. Senior and Richard W. Titball
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Medicine ,Science - Abstract
Galleria mellonella larvae are increasingly used to study the mechanisms of virulence of microbial pathogens and to assess the efficacy of antimicrobials. The G. mellonella model can faithfully reproduce many aspects of microbial disease which are seen in mammals, and therefore allows a reduction in the use of mammals. The model is now being widely used by researchers in universities, research institutes and industry. An attraction of the model is the interaction between pathogen and host. Hemocytes are specialised phagocytic cells which resemble neutrophils in mammals and play a major role in the response of the larvae to infection. However, the detailed interactions of hemocytes with pathogens is poorly understood, and is complicated by the presence of different sub-populations of cells. We report here a method for the isolation of hemocytes from Galleria mellonella. A needle-stick injury of larvae, before harvesting, markedly increased the recovery of hemocytes in the hemolymph. The majority of the hemocytes recovered were granulocyte-like cells. The hemocytes survived for at least 7 days in culture at either 28°C or 37°C. Pre-treatment of larvae with antibiotics did not enhance the survival of the cultured hemocytes. Our studies highlight the importance of including sham injected, rather than un-injected, controls when the G. mellonella model is used to test antimicrobial compounds. Our method will now allow investigations of the interactions of microbial pathogens with insect hemocytes enhancing the value of G. mellonella as an alternative model to replace the use of mammals, and for studies on hemocyte biology.
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- 2020
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12. Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity
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Helen Morcrette, Andrea Kovacs-Simon, Richard K. Tennant, John Love, Sariqa Wagley, Zheng R. Yang, David J. Studholme, Orkun S. Soyer, Olivia L. Champion, Clive S. Butler, and Richard W. Titball
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Campylobacter jejuni ,proteomics ,persister cell ,antibiotic ,electron transport ,Microbiology ,QR1-502 - Abstract
The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.
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- 2020
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13. Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction
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Siroj Jitprasutwit, Niramol Jitprasutwit, Claudia M. Hemsley, Nattawat Onlamoon, Patoo Withatanung, Veerachat Muangsombut, Paiboon Vattanaviboon, Joanne M. Stevens, Catherine Ong, Mark P. Stevens, Richard W. Titball, and Sunee Korbsrisate
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Burkholderia pseudomallei ,intracellular ,macrophage ,gene expression ,promoter trap library ,screen ,Microbiology ,QR1-502 - Abstract
Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5′ end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.
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- 2020
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14. Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy
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Rosemary A. Bamford, Ashley Smith, Jeremy Metz, Georgina Glover, Richard W. Titball, and Stefano Pagliara
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Viable but non-culturable cells ,Microfluidics ,Escherichia coli ,Persisters ,Single-cell ,Antibiotic tolerance ,Biology (General) ,QH301-705.5 - Abstract
Abstract Background Clonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population’s resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment. Methods Here, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment. Results We show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism. Conclusion Our data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.
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- 2017
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15. Correlating Genotyping Data of Coxiella burnetii with Genomic Groups
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Claudia M. Hemsley, Angela Essex-Lopresti, Isobel H. Norville, and Richard W. Titball
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Coxiella burnetii ,genotyping ,MST ,MLVA ,genomic groups ,Medicine - Abstract
Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. Several distinct genetic lineages or genomic groups have been shown to exist, with evidence for different virulence potential of these lineages. Multispacer Sequence Typing (MST) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) are being used to genotype strains. However, it is unclear how these typing schemes correlate with each other or with the classification into different genomic groups. Here, we created extensive databases for published MLVA and MST genotypes of C. burnetii and analysed the associated metadata, revealing associations between animal host and human disease type. We established a new classification scheme that assigns both MST and MLVA genotypes to a genomic group and which revealed additional sub-lineages in two genomic groups. Finally, we report a novel, rapid genomotyping method for assigning an isolate into a genomic group based on the Cox51 spacer sequence. We conclude that by pooling and streamlining existing datasets, associations between genotype and clinical outcome or host source were identified, which in combination with our novel genomotyping method, should enable an estimation of the disease potential of new C. burnetii isolates.
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- 2021
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16. An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions
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Nicola J. Senior, Kalesh Sasidharan, Richard J. Saint, Andrew E. Scott, Mitali Sarkar-Tyson, Philip M. Ireland, Helen L Bullifent, Z. Rong Yang, Karen Moore, Petra C. F. Oyston, Timothy P. Atkins, Helen S. Atkins, Orkun S. Soyer, and Richard W. Titball
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Yersinia pestis ,Plague ,TRADIS ,Transposon ,Essential genes ,Metabolic model ,Microbiology ,QR1-502 - Abstract
Abstract Background The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. Results Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. Conclusions Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.
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- 2017
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17. Interactions Between Pseudomonas Immunotoxins and the Plasma Membrane: Implications for CAT-8015 Immunotoxin Therapy
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Monika Bokori-Brown, Jeremy Metz, Peter G. Petrov, Francis Mussai, Carmela De Santo, Neil J. Smart, Sarah Saunders, Bridget Knight, Ira Pastan, Richard W. Titball, and C. Peter Winlove
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immunotoxin ,cancer treatment ,pediatric acute lymphoblastic leukemia ,CAT-8015 ,Moxetumomab pasudotox ,red blood cells ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Acute Lymphoblastic Leukemia (ALL) remains the most frequent cause of cancer-related mortality in children and novel therapies are needed for the treatment of relapsed/refractory childhood ALL. One approach is the targeting of ALL blasts with the Pseudomonas immunotoxin CAT-8015. Although CAT-8015 has potent anti-leukemia activity, with a 32% objective response rate in a phase 1 study of childhood ALL, haemolytic-uremic syndrome (HUS) and vascular leak syndrome (VLS), major dose-limiting toxicities, have limited the use of this therapeutic approach in children. Investigations into the pathogenesis of CAT-8015-induced HUS/VLS are hindered by the lack of an adequate model system that replicates clinical manifestations, but damage to vascular endothelial cells (ECs) and blood cells are believed to be major initiating factors in both syndromes. Since there is little evidence that murine models replicate human HUS/VLS, and CAT-8015-induced HUS/VLS predominantly affects children, we developed human models and used novel methodologies to investigate CAT-8015 interactions with red blood cells (RBCs) from pediatric ALL patients and ECs of excised human mesenteric arteries. We provide evidence that CAT-8015 directly interacts with RBCs, mediated by Pseudomonas toxin. We also show correlation between the electrical properties of the RBC membrane and RBC susceptibility to CAT-8015-induced lysis, which may have clinical implication. Finally, we provide evidence that CAT-8015 is directly cytototoxic to ECs of excised human mesenteric arteries. In conclusion, the human models we developed constitutes the first, and very important, step in understanding the origins of HUS/VLS in immunotoxin therapy and will allow further investigations of HUS/VLS pathogenesis.
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- 2018
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18. Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein
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Monika Bokori-Brown, Thomas G. Martin, Claire E. Naylor, Ajit K. Basak, Richard W. Titball, and Christos G. Savva
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Science - Abstract
Lysenin is member of the aerolysin family of small ß-barrel pore-forming toxins that include virulence factors from several human and animal pathogens. Here the authors determine the structure of the lysenin pore by single particle cryo- EM and propose a conserved pore formation mechanism for the aerolysin protein family.
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- 2016
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19. The Culture Environment Influences Both Gene Regulation and Phenotypic Heterogeneity in Escherichia coli
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Ashley Smith, Agnieszka Kaczmar, Rosemary A. Bamford, Christopher Smith, Simona Frustaci, Andrea Kovacs-Simon, Paul O’Neill, Karen Moore, Konrad Paszkiewicz, Richard W. Titball, and Stefano Pagliara
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phenotypic heterogeneity ,Escherichia coli ,persisters ,metabolism ,bacterial physiology ,antibiotics ,Microbiology ,QR1-502 - Abstract
Microorganisms shape the composition of the medium they are growing in, which in turn has profound consequences on the reprogramming of the population gene-expression profile. In this paper, we investigate the progressive changes in pH and sugar availability in the medium of a growing Escherichia coli (E. coli) culture. We show how these changes have an effect on both the cellular heterogeneity within the microbial community and the gene-expression profile of the microbial population. We measure the changes in gene-expression as E. coli moves from lag, to exponential, and finally into stationary phase. We found that pathways linked to the changes in the medium composition such as ribosomal, tricarboxylic acid cycle (TCA), transport, and metabolism pathways are strongly regulated during the different growth phases. In order to quantify the corresponding temporal changes in the population heterogeneity, we measure the fraction of E. coli persisters surviving different antibiotic treatments during the various phases of growth. We show that the composition of the medium in which β-lactams or quinolones, but not aminoglycosides, are dissolved strongly affects the measured phenotypic heterogeneity within the culture. Our findings contribute to a better understanding on how the composition of the culture medium influences both the reprogramming in the population gene-expression and the emergence of phenotypic variants.
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- 2018
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20. Identification of Possible Virulence Marker from Campylobacter jejuni Isolates
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James W. Harrison, Tran Thi Ngoc Dung, Fariha Siddiqui, Sunee Korbrisate, Habib Bukhari, My Phan Vu Tra, Nguyen Van Minh Hoang, Juan Carrique-Mas, Juliet Bryant, James I. Campbell, David J. Studholme, Brendan W. Wren, Stephen Baker, Richard W. Titball, and Olivia L. Champion
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Campylobacter jejuni ,Asia ,chicken ,type-six secretion system ,food security ,enteric infections ,Medicine ,Infectious and parasitic diseases ,RC109-216 - Abstract
A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.
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- 2014
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21. Identification of a Key Residue for Oligomerisation and Pore-Formation of Clostridium perfringens NetB
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Sérgio P. Fernandes da Costa, Christos G. Savva, Monika Bokori-Brown, Claire E. Naylor, David S. Moss, Ajit K. Basak, and Richard W. Titball
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NetB ,pore-forming toxin ,Clostridium perfringens ,necrotic enteritis ,Medicine - Abstract
Necrotic enteritis toxin B (NetB) is a β-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.
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- 2014
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22. Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens
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Olivia L. Champion, Richard W. Titball, and Steven Bates
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Galleria mellonella ,infection model ,fungi ,genome ,pathological score ,end point ,Biology (General) ,QH301-705.5 - Abstract
In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model. However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures. Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens. The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains. It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle.
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- 2018
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23. Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
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Madeleine G. Moule, Claudia M. Hemsley, Qihui Seet, José Afonso Guerra-Assunção, Jiali Lim, Mitali Sarkar-Tyson, Taane G. Clark, Patrick B. O. Tan, Richard W. Titball, Jon Cuccui, and Brendan W. Wren
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Microbiology ,QR1-502 - Abstract
ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
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- 2014
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24. Protection against Experimental Melioidosis following Immunisation with a Lipopolysaccharide-Protein Conjugate
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Andrew E. Scott, Sarah A. Ngugi, Thomas R. Laws, David Corser, Claire L. Lonsdale, Riccardo V. D’Elia, Richard W. Titball, E. Diane Williamson, Timothy P. Atkins, and Joann L. Prior
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Immunologic diseases. Allergy ,RC581-607 - Abstract
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.
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- 2014
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25. Correction: Melioidosis Vaccines: A Systematic Review and Appraisal of the Potential to Exploit Biodefense Vaccines for Public Health Purposes.
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Sharon J. Peacock, Direk Limmathurotsakul, Yoel Lubell, Gavin C. K. W. Koh, Lisa J. White, Nicholas P. J. Day, and Richard W. Titball
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Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Published
- 2013
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26. Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata
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Victória Valente Califre de Mello, Ana Julia Vidal Placa, Daniel Antonio Braga Lee, Eliz Oliveira Franco, Luciana Lima, Marta M.G. Teixeira, Claudia Hemsley, Richard W. Titball, Rosangela Zacarias Machado, and Marcos Rogério André
- Subjects
Infectious Diseases ,Insect Science ,Veterinary (miscellaneous) ,Parasitology - Published
- 2023
27. Molecular Survey of Anaplasmataceae Agents and Coxiellaceae in Non-Hematophagous Bats and Associated Ectoparasites from Brazil
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Marcos Rogério André, Carina Elisei de Oliveira, Rosangela Zacarias Machado, Heitor Miraglia Herrera, Priscila Ikeda, Richard W. Titball, Victória Valente Califre de Mello, Ana Julia Vidal Placa, Elizabete Captivo Lourenço, Claudia M. Hemsley, and Jaire Marinho Torres
- Subjects
Neorickettsia ,biology ,Ehrlichia ,Neorickettsia risticii ,Tick ,biology.organism_classification ,Ehrlichia ruminantium ,Anaplasma phagocytophilum ,Anaplasmataceae ,Coxiella burnetii ,Microbiology ,Ehrlichia spp ,Neorickettsia spp ,Chiroptera ,Anaplasma spp ,Anaplasma - Abstract
The Anaplasmataceae family (order Rickettsiales) encompasses obligately intracellular bacteria of the genera Anaplasma, Ehrlichia, and Neorickettsia. Together with Coxiella burnetii (Coxiellaceae family, order Legionellales), these bacteria represent important causative agents of diseases in humans and animals. The scarcity of studies that investigated the occurrence of these agents in bats and their associated ectoparasites, emphasizes the need to achieve a better understanding of the role of these animals in the maintenance of such bacteria. Herein, 418 samples (133 blood, 135 spleen, and 150 ectoparasites) are collected from 135 non-hematophagous bats belonging to 12 species in a periurban area of Campo Grande city, Mato Grosso do Sul state, midwestern Brazil. In the results, 1.65% (7/418), 12.04% (50/418), and 13.63% (57/418) of samples are positive in PCR assays for Anaplasma spp. (16S rRNA gene), Ehrlichia spp. (dsb gene), and Neorickettsia spp. (16S rRNA gene), respectively. Anaplasma spp. and Neorickettsia spp. are detected in one (5.26%) Ornithodoros hasei tick larva. Ehrlichia spp. is detected in 14% of bat flies (represented by Megistopoda aranea, Trichobius costalimai, and Strebla hertigi), 6% of tick larvae (O. hasei), 12% of Spinturnicidae mites (represented by Periglischrus sp., P. torrealbai, and P. acutisternus), and 38% of Macronyssidae mites (Steatonyssuss sp.). The obtained sequences are observed to be similar to Anaplasma phagocytophilum (97.42–97.6% identified), Ehrlichia minasensis (96.73–100% identified), Neorickettsia risticii (96.7–100% identified), and Neorickettsia findlayensis (95.07–100% identified) by BLASTn analyses, and closely related to Ehrlichia ruminantium by phylogenetic analyses based on the gltA gene. No bat samples (blood/spleen) are positive in the qPCR assay for C. burnetii based on the IS1111 gene. The present work shows, for the first time, the occurrence of Anaplasmataceae in bats and associated ectoparasites (ticks, mites, and bat flies) from Brazil.
- Published
- 2021
28. From protein microarrays to diagnostic antigen discovery: a study of the pathogen Francisella tularensis.
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Suman Sundaresh, Arlo Z. Randall, Berkay Unal, Jeannine M. Petersen, John T. Belisle, M. Gill Hartley, Melanie Duffield, Richard W. Titball, D. Huw Davies, Philip L. Felgner, and Pierre Baldi
- Published
- 2007
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29. Coxiella burnetii replicates in Galleria mellonella hemocytes and transcriptome mapping reveals in vivo regulated genes
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Isobel H. Norville, Georgie Metters, Claudia M. Hemsley, Richard W. Titball, and Andrea Kovacs-Simon
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Microbiology (medical) ,animal structures ,Immunology ,Virulence ,Vacuole ,Infectious and parasitic diseases ,RC109-216 ,Microbiology ,Transcriptome ,03 medical and health sciences ,Pathogen ,030304 developmental biology ,coxiella burnetii ,galleria mellonella ,0303 health sciences ,biology ,030306 microbiology ,Effector ,fungi ,transcriptome profiling ,biology.organism_classification ,Coxiella burnetii ,t4ss ,Galleria mellonella ,virulence ,Infectious Diseases ,Parasitology ,Bacteria ,Research Article ,Research Paper - Abstract
Larvae of the greater wax moth (Galleria mellonella) are susceptible to infection with C. burnetii, an obligate intracellular bacterial pathogen. We show that bacteria are found in hemocytes after infection, and occupy vacuoles which are morphologically similar to Coxiella-containing vacuoles seen in infected mammalian phagocytes. We characterized the infection by transcriptome profiling of bacteria isolated from the hemocytes of infected larvae and identified 46 highly upregulated genes. The encoded proteins are predicted to be involved in translation, LPS biosynthesis, biotin synthesis, scavenging of reactive oxygen species, and included a T4SS effector and 30 hypothetical proteins. Some of these genes had previously been shown to be upregulated in buffalo green monkey (BGM) cells or in mice, whilst others appear to be regulated in a host-specific manner. Altogether, our results demonstrate the value of the G. mellonella model to study intracellular growth and identify potential virulence factors of C. burnetii.
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- 2020
30. MLVA and com1 genotyping of Coxiella burnetii in farmed ruminants in Great Britain
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Claudia M. Hemsley, Angela Essex-Lopresti, Tom Chisnall, Mick Millar, Sue Neale, Rudolf Reichel, Isobel H. Norville, and Richard W. Titball
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General Veterinary ,General Medicine ,Microbiology - Abstract
Coxiella burnetii, the causative agent of the zoonotic disease Q fever, has been shown to be endemic in Great Britain, but information on the prevailing genomic lineages or Genomic Groups (GGs) of Coxiella burnetii is limited. The aim of this study was to genotype C. burnetii isolates from infected farmed ruminants by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) and identify their associated Genomic Group. A total of 51 Coxiella-containing abortion samples from farmed ruminants (sheep, goats, and cattle), which were collected in Great Britain during 2013-2018, were included in the study, 34 of which returned a C. burnetii MLVA genotype. All bovine samples (n = 18), 5/7 of the ovine samples, and 3/9 of the caprine samples belonged to an MLVA cluster which we could link to the MST20 genotype of GG III, whereas 6/9 of the caprine samples and 2/7 of the ovine samples belonged to MLVA clusters which we could link to the MST33 or MST32 genotypes of GG II (7 vs 1 sample(s), respectively). We also noted that the Coxiella-specific com1 gene contained unique mutations that could genomotype isolates, i.e. assign them to a Genomic Group. In conclusion, both goats and sheep in Great Britain (from 2014 onward) were found to carry the same MLVA genotypes (MST33-like; GG II) that were linked to a human Q fever outbreak in the Netherlands. This knowledge in combination with the usage of genotyping/genomotyping methods should prove useful in future surveillance programs and in the management of outbreaks.
- Published
- 2022
31. A genetic programming approach for Burkholderia Pseudomallei diagnostic pattern discovery.
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Zheng Rong Yang, Ganjana Lertmemongkolchai, Gladys Tan, Philip L. Felgner, and Richard W. Titball
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- 2009
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32. From cell culture to cynomolgus macaque: infection models show lineage-specific virulence potential of Coxiella burnetii
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Isobel H. Norville, Georgina Metters, Richard W. Titball, and Claudia M. Hemsley
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0301 basic medicine ,Microbiology (medical) ,Obligate ,biology ,030106 microbiology ,Virulence ,Q fever ,General Medicine ,Coxiella burnetii ,biology.organism_classification ,medicine.disease ,Microbiology ,03 medical and health sciences ,030104 developmental biology ,Cell culture ,medicine ,Pathogen ,Intracellular ,Bacteria - Abstract
Coxiella burnetii is an obligate intracellular pathogen that causes the zoonotic disease Q fever in humans, which can occur in either an acute or a chronic form with serious complications. The bacterium has a wide host range, including unicellular organisms, invertebrates, birds and mammals, with livestock representing the most significant reservoir for human infections. Cell culture models have been used to decipher the intracellular lifestyle of C. burnetii, and several infection models, including invertebrates, rodents and non-human primates, are being used to investigate host-pathogen interactions and to identify bacterial virulence factors and vaccine candidates. However, none of the models replicate all aspects of human disease. Furthermore, it is becoming evident that C. burnetii isolates belonging to different lineages exhibit differences in their virulence in these models. Here, we compare the advantages and disadvantages of commonly used infection models and summarize currently available data for lineage-specific virulence.
- Published
- 2019
33. Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein
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Leo Bennett, Helen Morcrette, Monika Bokori-Brown, Brendan W. Wren, Richard W. Titball, Stephanie Ong, and N. Lewis
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lcsh:Immunologic diseases. Allergy ,Protein vaccines ,Lymphocyte ,Immunology ,medicine.disease_cause ,lcsh:RC254-282 ,Article ,03 medical and health sciences ,Myelin ,parasitic diseases ,medicine ,Pharmacology (medical) ,Receptor ,030304 developmental biology ,Pharmacology ,0303 health sciences ,biology ,030306 microbiology ,Chemistry ,Toxin ,Chinese hamster ovary cell ,Clostridium perfringens epsilon toxin ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Molecular biology ,3. Good health ,Infectious Diseases ,medicine.anatomical_structure ,Toxicity ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Antibody ,Bacterial infection ,lcsh:RC581-607 - Abstract
A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin–Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine., Genetic toxoids avoid MAL for reduced host toxicity Cells expressing myelin and lymphocyte protein (MAL), the putative receptor for Clostridium perfringens’ epsilon toxin, can be sensitive to otherwise attenuated mutants of the toxin. Here, the team led by Richard Titball at United Kingdom’s University of Exeter found that a previous variant exhibits differential toxic effects when cells express sheep or human MAL. To circumvent this, Titball’s team applied site-directed mutagenesis of the receptor binding site to develop a new variant with enhanced reduction in toxicity towards MAL-expressing cells and able to induce high levels of neutralising antibodies upon immunisation of sheep. These findings suggests that testing genetic toxoids in cells expressing MAL from the target species might be relevant for enterotoxaemia vaccine development and warrant further studies into the role of MAL in epsilon toxin-mediated pathogenesis.
- Published
- 2019
34. Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages
- Author
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Richard W. Titball, Joann L. Prior, Andrew E. Scott, Andrea Kovacs-Simon, and Claudia M. Hemsley
- Subjects
Microbiology (medical) ,Burkholderia pseudomallei ,Proteome ,Burkholderia ,Virulence Factors ,lcsh:QR1-502 ,Virulence ,Human pathogen ,Microbiology ,Genome ,lcsh:Microbiology ,Cell Line ,03 medical and health sciences ,Mice ,Animals ,Gene ,0303 health sciences ,Microbial Viability ,biology ,Strain (chemistry) ,Burkholderia thailandensis ,Whole Genome Sequencing ,030306 microbiology ,Gene Expression Profiling ,Macrophages ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,bacterial infections and mycoses ,3. Good health ,Parasitology ,Host-Pathogen Interactions ,bacteria ,Burkholderia thailandensis strain E555 ,Transcriptome ,Genome, Bacterial ,Research Article - Abstract
Background Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. Results We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. Conclusions A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms. Electronic supplementary material The online version of this article (10.1186/s12866-019-1469-8) contains supplementary material, which is available to authorized users.
- Published
- 2019
35. Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model
- Author
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Timothy Vartanian, Monika Bokori-Brown, Samantha V. Shetty, Drew Adler, Yinghua Ma, Richard W. Titball, and Jennifer R. Linden
- Subjects
0301 basic medicine ,animal structures ,ved/biology.organism_classification_rank.species ,02 engineering and technology ,Vascular Remodeling ,Blood–brain barrier ,Article ,Pathogenic Organism ,03 medical and health sciences ,Myelin ,Downregulation and upregulation ,medicine ,lcsh:Science ,Receptor ,Model organism ,Zebrafish ,Multidisciplinary ,Model Organism ,biology ,ved/biology ,Chemistry ,Clostridium perfringens epsilon toxin ,021001 nanoscience & nanotechnology ,biology.organism_classification ,3. Good health ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,cardiovascular system ,lcsh:Q ,Molecular Mechanism of Behavior ,0210 nano-technology ,Blood vessel - Abstract
Summary Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor., Graphical Abstract, Highlights • ProETX binds specifically to hMAL in live humanized zebrafish • hMAL expression in zebrafish confers susceptibility to ETX-mediated BBB breakdown • Live imaging reveals ETX-mediated edema, hMAL downregulation, stenosis, and stasis • Antibody neutralization abrogates ETX-mediated vascular pathology, Pathogenic Organism; Vascular Remodeling; Molecular Mechanism of Behavior; Model Organism
- Published
- 2019
36. Novel multi-component vaccine approaches for Burkholderia pseudomallei
- Author
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Richard W. Titball, Lisa A. Morici, and Alfredo G. Torres
- Subjects
0301 basic medicine ,Burkholderia pseudomallei ,Melioidosis ,Immunology ,Virulence ,CD8-Positive T-Lymphocytes ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Antigen ,Immunity ,medicine ,Animals ,Humans ,Immunology and Allergy ,Pathogen ,Antigens, Bacterial ,Attenuated vaccine ,Vaccines for Emerging Pathogens: from Research to the Clinic. Part 1. Series Editor: E Diane Williamson ,biology.organism_classification ,medicine.disease ,Antibodies, Bacterial ,Virology ,030104 developmental biology ,Bacterial Vaccines ,030215 immunology - Abstract
Summary Burkholderia pseudomallei is the causative agent of melioidosis. Historically believed to be a relatively rare human disease in tropical countries, a recent study estimated that, worldwide, there are approximately 165 000 human melioidosis cases per year, more than half of whom die. The bacterium is inherently resistant to many antibiotics and treatment of the disease is often protracted and ineffective. There is no licensed vaccine against melioidosis, but a vaccine is predicted to be of value if used in high-risk populations. There has been progress over the last decade in the pursuit of an effective vaccine against melioidosis. Animal models of disease including mouse and non-human primates have been developed, and these models show that antibody responses play a key role in protection against melioidosis. Surprisingly, although B. pseudomallei is an intracellular pathogen there is limited evidence that CD8+ T cells play a role in protection. It is evident that a multi-component vaccine, incorporating one or more protective antigens, will probably be essential for protection because of the pathogen's sophisticated virulence mechanisms as well as strain heterogeneity. Multi-component vaccines in development include glycoconjugates, multivalent subunit preparations, outer membrane vesicles and other nano/microparticle platforms and live-attenuated or inactivated bacteria. A consistent finding with vaccine candidates tested in mice is the ability to induce sterilizing immunity at low challenge doses and extended time to death at higher challenge doses. Further research to identify ways of eliciting more potent immune responses might provide a path for licensing an effective vaccine.
- Published
- 2019
37. Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival
- Author
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Sariqa Wagley, John Love, Richard W. Titball, Zheng R. Yang, Richard K. Tennant, Clive S. Butler, Neil Murray, Ann Power, Helen Morcrette, and Andrea Kovacs-Simon
- Subjects
Proteomics ,Life Cycles ,Critical Care and Emergency Medicine ,Proteome ,Proteomes ,Protein Expression ,Biochemistry ,chemistry.chemical_compound ,Larvae ,Medicine and Health Sciences ,Electron Microscopy ,Biology (General) ,Cells, Cultured ,Staining ,Growth medium ,Microscopy ,Microbial Viability ,biology ,Virulence ,Vibrio parahaemolyticus ,Cell Staining ,Ketones ,Galleria mellonella ,Chemistry ,Physical Sciences ,Scanning Electron Microscopy ,Research Article ,Pyruvate ,QH301-705.5 ,Resuscitation ,Immunology ,Research and Analysis Methods ,Bacterial Physiological Phenomena ,Microbiology ,Downregulation and upregulation ,Bacterial Proteins ,Virology ,Lactate dehydrogenase ,Genetics ,Gene Expression and Vector Techniques ,Molecular Biology Techniques ,Gene ,Molecular Biology ,Molecular Biology Assays and Analysis Techniques ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Metabolism ,Gene Expression Regulation, Bacterial ,RC581-607 ,biology.organism_classification ,Culture Media ,chemistry ,Specimen Preparation and Treatment ,Vibrio Infections ,Dormancy ,Parasitology ,Immunologic diseases. Allergy ,Acids ,Developmental Biology - Abstract
The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state., Author summary Members of the Proteobacteria are reported to adopt a survival strategy and enter a viable but non culturable (VBNC) state, when exposed to stressful or non-permissible growth conditions. This is a characteristic employed widely in the natural environment in order for bacteria to survive harsh environmental conditions over a long period. In spite of the importance of the VBNC state in microbiology, we know little about the molecular makeup of VBNC cells. In this study, using the model organism Vibrio parahaemolyticus, we resolved that distinct subpopulations of bacteria exist in the VBNC state and these have different resuscitation potentials and distinct proteomic profiles. We also discovered that deletion of the gene encoding the enzyme lactate dehydrogenase (lldD) triggered the cells to enter the VBNC state, and adding lactate to VBNC cells extended their resuscitation potential window. The ability for bacteria to survive in the VBNC state might be linked to their ability to overcome oxidative stress.
- Published
- 2021
38. Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence
- Author
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Joanne M. Stevens, Metawee Thongdee, Somjit Chaiwattanarungruengpaisan, Chatruthai Meethai, Sunee Korbsrisate, Richard W. Titball, Varintip Srinon, and Patoo Withatanung
- Subjects
0301 basic medicine ,Burkholderia pseudomallei ,Virulence Factors ,Immunology ,030106 microbiology ,Mutant ,lcsh:Medicine ,Virulence ,medicine.disease_cause ,Microbiology ,Article ,Cell Line ,Mice ,03 medical and health sciences ,Bacterial Proteins ,Gene expression ,medicine ,Animals ,lcsh:Science ,Multidisciplinary ,biology ,Phospholipase C ,lcsh:R ,Pathogenic bacteria ,biology.organism_classification ,030104 developmental biology ,Melioidosis ,Cell culture ,Type C Phospholipases ,lcsh:Q ,Bacteria - Abstract
Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.
- Published
- 2020
39. Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity
- Author
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Richard K. Tennant, Sariqa Wagley, John Love, Andrea Kovacs-Simon, Orkun S. Soyer, Zheng R. Yang, Helen Morcrette, Olivia L. Champion, David J. Studholme, Clive S. Butler, and Richard W. Titball
- Subjects
0301 basic medicine ,Microbiology (medical) ,RM ,Multidrug tolerance ,medicine.drug_class ,030106 microbiology ,Immunology ,Population ,Antibiotics ,lcsh:QR1-502 ,Microbiology ,Campylobacter jejuni ,lcsh:Microbiology ,03 medical and health sciences ,Cellular and Infection Microbiology ,proteomics ,antibiotic ,medicine ,electron transport ,education ,Original Research ,education.field_of_study ,biology ,Chemistry ,QH ,Membrane hyperpolarization ,biology.organism_classification ,QP ,QR ,Penicillin ,persister cell ,030104 developmental biology ,Infectious Diseases ,Intracellular ,Bacteria ,medicine.drug - Abstract
The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.
- Published
- 2020
40. Trehalose and bacterial virulence
- Author
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Richard W. Titball and Muthita Vanaporn
- Subjects
Microbiology (medical) ,Immunology ,Defence mechanisms ,Virulence ,Infectious and parasitic diseases ,RC109-216 ,Review Article ,Biology ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Stress, Physiological ,Animals ,Humans ,Pathogen ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Bacteria ,030306 microbiology ,Abiotic stress ,Trehalose ,Glycosidic bond ,Bacterial Infections ,Plants ,biology.organism_classification ,Bacterial vaccine ,virulence ,Infectious Diseases ,chemistry ,Biochemistry ,Host-Pathogen Interactions ,Parasitology ,pathogen - Abstract
Trehalose is a disaccharide of two D-glucose molecules linked by a glycosidic linkage, which plays both structural and functional roles in bacteria. Trehalose can be synthesized and degraded by several pathways, and induction of trehalose biosynthesis is typically associated with exposure to abiotic stress. The ability of trehalose to protect against abiotic stress has been exploited to stabilize a range of bacterial vaccines. More recently, there has been interest in the role of this molecule in microbial virulence. There is now evidence that trehalose or trehalose derivatives play important roles in virulence of a diverse range of Gram-positive and Gram-negative pathogens of animals or plants. Trehalose and/or trehalose derivatives can play important roles in host colonization and growth in the host, and can modulate the interactions with host defense mechanisms. However, the roles are typically pathogen-specific. These findings suggest that trehalose metabolism may be a target for novel pathogen-specific rather than broad spectrum interventions.
- Published
- 2020
41. Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction
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Veerachat Muangsombut, Niramol Jitprasutwit, Mark P. Stevens, Joanne M. Stevens, Siroj Jitprasutwit, Nattawat Onlamoon, Paiboon Vattanaviboon, Sunee Korbsrisate, Catherine Ong, Claudia M. Hemsley, Richard W. Titball, and Patoo Withatanung
- Subjects
Microbiology (medical) ,Burkholderia pseudomallei ,Melioidosis ,lcsh:QR1-502 ,screen ,Virulence ,macrophage ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,Plasmid ,medicine ,differential fluorescence induction ,Gene ,Original Research ,030304 developmental biology ,0303 health sciences ,biology ,Burkholderia thailandensis ,030306 microbiology ,Promoter ,biology.organism_classification ,medicine.disease ,bacterial infections and mycoses ,intracellular ,3. Good health ,genomic DNA ,promoter trap library ,gene expression ,bacteria - Abstract
Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.
- Published
- 2020
42. Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis
- Author
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Andrea Malaspina, Dominika Raciborska, Ben Turner, Richard W. Titball, Helen Morcrette, Sharmilee Gnanapavan, Michel R. Popoff, Richard Nicholas, N. Lewis, Sariqa Wagley, Monika Bokori-Brown, Caroline D’Arcy, College of Life and Environmental Sciences [Exeter], University of Exeter, Queen Mary University of London (QMUL), West London Neuroscience Centre [Charing Cross Hospital, London], Charing Cross Hospital & Imperial College, MS sciences limited, Bactéries anaérobies et Toxines, Institut Pasteur [Paris], Division of Brain Sciences [London], Imperial College London, Barts Health NHS Trust [London, UK], The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by the MS Sciences Ltd and by the NIHR Exeter CRF., and Institut Pasteur [Paris] (IP)
- Subjects
Multiple Sclerosis ,Clostridium perfringens ,[SDV]Life Sciences [q-bio] ,Bacterial Toxins ,epsilon toxin ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,[SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] ,CHO Cells ,medicine.disease_cause ,03 medical and health sciences ,Cricetulus ,0302 clinical medicine ,[SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB] ,medicine ,Animals ,Humans ,030212 general & internal medicine ,Clinically isolated syndrome ,biology ,business.industry ,Toxin ,Multiple sclerosis ,Clostridium perfringens epsilon toxin ,medicine.disease ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Molecular biology ,3. Good health ,Blot ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Neurology ,Pepscan ,biology.protein ,Neurology (clinical) ,Antibody ,business ,Original Research Papers ,030217 neurology & neurosurgery - Abstract
Background: It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease. Objective: We investigated for serum antibodies against Etx in UK patients with clinically definite multiple sclerosis (CDMS) or presenting with clinically isolated syndrome (CIS) or optic neuritis (ON) and in age- and gender-matched controls. Methods: We tested sera from CDMS, CIS or ON patients or controls by Western blotting. We also tested CDMS sera for reactivity with linear overlapping peptides spanning the amino acid sequence (Pepscan) of Etx. Results: Using Western blotting, 24% of sera in the combined CDMS, CIS and ON groups ( n = 125) reacted with Etx. In the control group ( n = 125), 10% of the samples reacted. Using Pepscan, 33% of sera tested reacted with at least one peptide, whereas in the control group only 16% of sera reacted. Out of 61 samples, 21 (43%) were positive to one or other testing methodology. Three samples were positive by Western blotting and Pepscan. Conclusion: Our results broadly support the previous findings and the role of Etx in the aetiology of MS warrants further investigation.
- Published
- 2018
43. Galleria mellonella as an infection model to investigate virulence of Vibrio parahaemolyticus
- Author
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Jamie W. Harrison, Varaporn Vuddhakul, Richard Borne, Richard W. Titball, Craig Baker-Austin, Donatella Ottaviani, Sariqa Wagley, and Francesca Leoni
- Subjects
0301 basic medicine ,Microbiology (medical) ,endocrine system ,animal structures ,030106 microbiology ,Immunology ,MutT ,Virulence ,Microbiology ,Nudix hydrolase ,lcsh:Infectious and parasitic diseases ,03 medical and health sciences ,nudix hydrolase ,lcsh:RC109-216 ,biology ,Vibrio parahaemolyticus ,fungi ,food and beverages ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Virology ,3. Good health ,Galleria mellonella ,Infectious Diseases ,bacteria ,Parasitology - Abstract
Non-toxigenic V. parahaemolyticus isolates (tdh−/trh−/T3SS2−) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mellonella) are susceptible to infection by toxigenic or non-toxigenic clinical isolates of V. parahaemolyticus. In comparison larvae inoculated with environmental isolates of V. parahaemolyticus did not succumb to disease. Whole genome sequencing of clinical non-toxigenic isolates revealed the presence of a gene encoding a nudix hydrolase, identified as mutT. A V. parahaemolyticus mutT mutant was unable to kill G. mellonella at 24 h post inoculation, indicating a role of this gene in virulence. Our findings show that G. mellonella is a valuable model for investigating screening of possible virulence genes of V. parahaemolyticus and can provide new insights into mechanisms of virulence of atypical non-toxigenic V. parahaemolyticus. These findings will allow improved genetic tests for the identification of pathogenic V. parahaemolyticus to be developed and will have a significant impact for the scientific community.
- Published
- 2017
44. Correlating Genotyping Data of Coxiella burnetii with Genomic Groups
- Author
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Richard W. Titball, Isobel H. Norville, Angela E. Essex-Lopresti, and Claudia M. Hemsley
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Microbiology (medical) ,Virulence ,Q fever ,Multiple Loci VNTR Analysis ,Coxiella burnetii ,genotyping ,MST ,MLVA ,genomic groups ,Genotype ,medicine ,Immunology and Allergy ,Typing ,Molecular Biology ,Genotyping ,Genetics ,General Immunology and Microbiology ,biology ,bacterial infections and mycoses ,medicine.disease ,biology.organism_classification ,Variable number tandem repeat ,Infectious Diseases ,Medicine - Abstract
Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. Several distinct genetic lineages or genomic groups have been shown to exist, with evidence for different virulence potential of these lineages. Multispacer Sequence Typing (MST) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) are being used to genotype strains. However, it is unclear how these typing schemes correlate with each other or with the classification into different genomic groups. Here, we created extensive databases for published MLVA and MST genotypes of C. burnetii and analysed the associated metadata, revealing associations between animal host and human disease type. We established a new classification scheme that assigns both MST and MLVA genotypes to a genomic group and which revealed additional sub-lineages in two genomic groups. Finally, we report a novel, rapid genomotyping method for assigning an isolate into a genomic group based on the Cox51 spacer sequence. We conclude that by pooling and streamlining existing datasets, associations between genotype and clinical outcome or host source were identified, which in combination with our novel genomotyping method, should enable an estimation of the disease potential of new C. burnetii isolates.
- Published
- 2021
45. Burkholderia pseudomallei and Burkholderia mallei vaccines: Are we close to clinical trials?
- Author
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Richard W. Titball, Mary N. Burtnick, Paul J. Brett, and Gregory J. Bancroft
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0301 basic medicine ,Burkholderia pseudomallei ,Melioidosis ,Disease ,Biology ,Burkholderia mallei ,Microbiology ,03 medical and health sciences ,medicine ,Animals ,Humans ,Clinical Trials as Topic ,General Veterinary ,General Immunology and Microbiology ,Glanders ,Public Health, Environmental and Occupational Health ,medicine.disease ,biology.organism_classification ,Vaccine efficacy ,Virology ,Clinical trial ,030104 developmental biology ,Infectious Diseases ,Bacterial Vaccines ,Biological warfare ,Molecular Medicine - Abstract
B. pseudomallei is the cause of melioidosis, a serious an often fatal disease of humans and animals. The closely related bacterium B. mallei, which cases glanders, is considered to be a clonal derivative of B. pseudomallei. Both B. pseudomallei and B. mallei were evaluated by the United States and the former USSR as potential bioweapons. Much of the effort to devise biodefence vaccines in the past decade has been directed towards the identification and formulation of sub-unit vaccines which could protect against both melioidosis and glanders. A wide range of proteins and polysaccharides have been identified which protective immunity in mice. In this review we highlight the significant progress that has been made in developing glycoconjugates as sub-unit vaccines. We also consider some of the important the criteria for licensing, including the suitability of the "animal rule" for assessing vaccine efficacy, the protection required from a vaccine and the how correlates of protection will be identified. Vaccines developed for biodefence purposes could also be used in regions of the world where naturally occurring disease is endemic.
- Published
- 2017
46. From cell culture to cynomolgus macaque: infection models show lineage-specific virulence potential of
- Author
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Georgina, Metters, Isobel H, Norville, Richard W, Titball, and Claudia M, Hemsley
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Disease Models, Animal ,Macaca fascicularis ,Species Specificity ,Virulence ,Coxiella burnetii ,Animals ,Humans ,Q Fever ,Phylogeny - Published
- 2019
47. The global impact and cost-effectiveness of a melioidosis vaccine
- Author
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Direk Limmathurotsakul, Nantasit Luangasanatip, Bart J. Currie, Stefan Flasche, Chiranjay Mukhopadhyay, Tim P. Atkins, Mark Jit, David A. B. Dance, and Richard W. Titball
- Subjects
Marginal cost ,Male ,Melioidosis ,Cost effectiveness ,Cost-Benefit Analysis ,lcsh:Medicine ,Infections ,03 medical and health sciences ,0302 clinical medicine ,Per capita ,Medicine ,Humans ,030212 general & internal medicine ,health care economics and organizations ,business.industry ,Transmission (medicine) ,lcsh:R ,Vaccination ,General Medicine ,Middle Aged ,medicine.disease ,Economic evaluation ,3. Good health ,Cohort ,Cost-effectiveness ,Female ,business ,Vaccine ,030217 neurology & neurosurgery ,Demography ,Research Article - Abstract
Background Every year, 90,000 people may die from melioidosis. Vaccine candidates have not proceeded past animal studies, partly due to uncertainty around the potential market size. This study aims to estimate the potential impact, cost-effectiveness and market size for melioidosis vaccines. Methods Age-structured decision tree models with country-specific inputs were used to estimate net costs and health benefits of vaccination, with health measured in quality-adjusted life years (QALYs). Four target groups of people living in endemic regions were considered: (i) people aged over 45 years with chronic renal disease, (ii) people aged over 45 years with diabetes, (iii) people aged over 45 years with diabetes and/or chronic renal disease, (iv) everyone aged over 45 years. Melioidosis risk was estimated using Bayesian evidence synthesis of 12 observational studies. In the base case, vaccines were assumed to have 80% efficacy, to have 5-year mean protective duration and to cost USD10.20–338.20 per vaccine. Results Vaccination could be cost-effective (with incremental cost-effectiveness ratio below GDP per capita) in 61/83 countries/territories with local melioidosis transmission. In these 61 countries/territories, vaccination could avert 68,000 lost QALYs, 8300 cases and 4400 deaths per vaccinated age cohort, at an incremental cost of USD59.6 million. Strategy (ii) was optimal in most regions. The vaccine market may be worth USD268 million per year at its threshold cost-effective price in each country/territory. Conclusions There is a viable melioidosis vaccine market, with cost-effective vaccine strategies in most countries/territories with local transmission. Electronic supplementary material The online version of this article (10.1186/s12916-019-1358-x) contains supplementary material, which is available to authorized users.
- Published
- 2019
48. Quantitative Proteomics Reveals Differences in the Response of Neutrophils Isolated from Healthy or Diabetic Subjects to Infection with Capsule-Variant Burkholderia thailandensis
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Dominic Kurian, Watip Tangjittipokin, Richard W. Titball, Sunee Korbsrisate, Patoo Withatanung, Joanne M. Stevens, and Nattachet Plengvidhya
- Subjects
0301 basic medicine ,Proteomics ,Programmed cell death ,Melioidosis ,Burkholderia ,Neutrophils ,Burkholderia thailandensis ,Inflammation ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Diabetes mellitus ,medicine ,Diabetes Mellitus ,Humans ,Cells, Cultured ,030102 biochemistry & molecular biology ,biology ,Cell Death ,Burkholderia pseudomallei ,business.industry ,neutrophil ,Burkholderia Infections ,General Chemistry ,medicine.disease ,biology.organism_classification ,030104 developmental biology ,iTRAQ ,Case-Control Studies ,diabetes mellitus ,Immunology ,Cytokines ,Tumor necrosis factor alpha ,melioidosis ,medicine.symptom ,business ,Oxidative stress ,BTCV - Abstract
In Thailand, diabetes mellitus is the most significant risk factor for melioidosis, a severe disease caused by Burkholderia pseudomallei. In this study, neutrophils isolated from healthy or diabetic subjects were infected with B. thailandensis E555, a variant strain with a B. pseudomallei-like capsular polysaccharide used here as a surrogate micro-organism for B. pseudomallei. At 2 h post-infection, neutrophil proteins were subjected to 4-plex iTRAQ-based comparative proteomic analysis. A total of 341 proteins were identified in two or more samples, of which several proteins involved in oxidative stress and inflammation were enriched in infected diabetic neutrophils. We validated this finding by demonstrating that infected diabetic neutrophils generated significantly elevated levels of pro-inflammatory cytokines TNFα, IL-6, IL-1β, and IL-17 compared to healthy neutrophils. Our data also revealed that infected neutrophils from healthy or diabetic individuals undergo apoptotic cell death at distinctly different rates, with infected diabetic neutrophils showing a diminished ability to delay apoptosis and an increased likelihood of undergoing a lytic form of cell death, compared to infected neutrophils from healthy individuals. Increased expression of inflammatory proteins by infected neutrophils could contribute to the increased susceptibility to infection and inflammation in diabetic patients in melioidosis-endemic areas.
- Published
- 2019
49. Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups
- Author
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Angela E. Essex-Lopresti, Richard W. Titball, Tim P. Atkins, Claudia M. Hemsley, Isobel H. Norville, and Paul O'Neill
- Subjects
0106 biological sciences ,Genotyping ,lcsh:QH426-470 ,Genotyping Techniques ,lcsh:Biotechnology ,Single-nucleotide polymorphism ,Q fever ,Pan-Genome Analysis ,01 natural sciences ,Genome ,Evolution, Molecular ,03 medical and health sciences ,lcsh:TP248.13-248.65 ,Genotype ,Genetics ,medicine ,Animals ,Phylogeny ,030304 developmental biology ,2. Zero hunger ,Whole genome sequencing ,0303 health sciences ,Genetic diversity ,Patho-adaptation ,biology ,Whole Genome Sequencing ,Genomics ,Coxiella burnetii ,biology.organism_classification ,medicine.disease ,bacterial infections and mycoses ,United Kingdom ,3. Good health ,lcsh:Genetics ,bacteria ,Cattle ,Genome, Bacterial ,010606 plant biology & botany ,Biotechnology ,Research Article ,Genome-Wide Association Study - Abstract
Background Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. Results Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. Conclusions The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen. Electronic supplementary material The online version of this article (10.1186/s12864-019-5833-8) contains supplementary material, which is available to authorized users.
- Published
- 2019
50. Global Analysis of Genes Essential for Francisella tularensis Schu S4 Growth In Vitro and for Fitness during Competitive Infection of Fischer 344 Rats
- Author
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Rachel E. Ireland, Richard W. Titball, Philip M. Ireland, Helen L. Bullifent, Nicola J. Senior, Zheng Rong Yang, Stephanie J. Southern, Andrew E. Scott, Michelle Nelson, and Helen S. Atkins
- Subjects
Transposable element ,Virulence Factors ,DNA Mutational Analysis ,Neocallimastigales ,Virulence ,Microbiology ,Tularemia ,transposon insertion sequencing ,03 medical and health sciences ,genomics ,medicine ,essential genes ,Animals ,Genetic Testing ,Francisella ,Francisella tularensis ,Molecular Biology ,Gene ,Pathogen ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Infectious dose ,biology.organism_classification ,medicine.disease ,Rats, Inbred F344 ,virulence ,Disease Models, Animal ,Mutagenesis, Insertional ,Genes, Bacterial ,gene essentiality ,TraDIS ,DNA Transposable Elements ,Research Article - Abstract
The intracellular bacterial pathogen Francisella tularensis causes a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches. F. tularensis is one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important for F. tularensis under in vitro and in vivo conditions, providing candidates that can be evaluated for vaccine or antibacterial development., The highly virulent intracellular pathogen Francisella tularensis is a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis of Francisella virulence in the Fischer 344 rat, we have constructed an F. tularensis Schu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growth in vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. This in vivo selection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-type F. tularensis Schu S4 strain. IMPORTANCE The intracellular bacterial pathogen Francisella tularensis causes a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches. F. tularensis is one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important for F. tularensis under in vitro and in vivo conditions, providing candidates that can be evaluated for vaccine or antibacterial development.
- Published
- 2019
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