- Volume 2, Issue 7A, 2020
Volume 2, Issue 7A, 2020
- Abstracts from Annual Conference 2020
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- Poster Presentation
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Structural investigation of cyclic nucleotide binding proteins from Trypanosoma cruzi
More LessFora targeted therapy of Trypanosomiasis, new antiparasitic drugs should be specifically directed against essential pathways in the parasite life cycle. Among these potential targets are signal transduction pathways, which have remained largely unexplored in Trypanosoma species. Of special interest is cAMP-mediated signaling, since cAMP has been shown to play critical roles in the life cycle of T. cruzi and in host cell during invasion. The presented research focuses on the identification and characterisation of novel cAMP response proteins (CARPs) in T. cruzi by using a multi disciplinary approach involving the parasitology group of Dr Martin Edreira (University of Buenos Aires, Argentina) and the structural biology group of Dr Ivan Campeotto (University of Leicester, UK). The aim of the project is not only to increase our knowledge about T. cruzi biology but also to target CARPs for the design and development of novel therapeutic agents against Chagas disease. To date, protein crystals of one of the members of the CARP family have been obtained, paving the way for structure determination and for a structure-based drug design approach.
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Diverse mutational routes to the restoration of motility in a soil bacterium
More LessRestoration of a phenotype following insertion of a crippling mutation gives us insights into the adaptability of an organism. Mutational routes to the restoration of phenotypes can be diverse. Deletion of the master regulator (fleQ) for flagellar synthesis genes renders Pseudomonas fluorescens immotile, but after being put under strong selection to swim, it reliably re-evolves motility.
In order to uncover how the immotile bacteria regain their ability to swim they are inoculated into swimming agar plates and sampled from the leading edge of the motile zone. These samples are sequenced at a range single locus sites, predicted to be where causative mutations for the restoration of motility will be found (as indicated from previous research).
In the engineered immotile strain Pf0-2x, a huge variety of mutations, consisting of SNPs, multi-codon deletions, and frameshifts are observed across all candidate loci. In a complex environment (LB) 50% of initial restorative mutations occur in glnA, a glutamine synthetase gene. In a minimal media environment (M9) 45% of these mutations occur in ntrB, a gene for a kinase that forms part of a two component system. These findings contrast strongly with similar work in the related P. fluorescens strain SBW25, where the same SNP is repeatedly found across all environments. Interestingly, this SNP has never been seen in Pf0-2x.
These results highlight how preferential mutational routes can vary across environments, and that strain-to-strain differences can have a major impact types of mutations that are uncovered and selected for by evolution.
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Genomic epidemiology of Campylobacter jejuni associated with asymptomatic pediatric infection in the Peruvian Amazon
More LessCampylobacter is the leading bacterial cause of gastroenteritis worldwide and its incidence is especially high in low- and middle-income countries (LMIC). Disease epidemiology in LMICs is different compared to high income countries like the USA or in Europe. Children in LMICs commonly have repeated and chronic infections even in the absence of symptoms, which can lead to deficits in early childhood development. In this study, we sequenced and characterized C. jejuni (n=62) from a longitudinal cohort study of children under the age of 5 with and without diarrheal symptoms, and contextualized them within a global C. jejuni genome collection. Epidemiological differences in disease presentation were reflected in the genomes, specifically by the absence of some of the most common global disease-causing lineages. As in many other countries, poultry-associated strains were a major source of human infection but almost half of local disease cases (15 of 31) were attributable to genotypes that are rare outside of Peru. Asymptomatic infection was not limited to a single (or few) human adapted lineages but resulted from phylogenetically divergent strains suggesting an important role for host factors in the cryptic epidemiology of campylobacteriosis in LMICs. Preprint: https://www.medrxiv.org/content/10.1101/2020.04.26.20075689v1
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Antimicrobial resistance: Transdisciplinary research on humans, antimicrobials and microbes
More LessCalls for action on antimicrobial resistance (AMR) have existed almost as early as the discovery of penicillin and the sulpha drugs. Since then solutions to AMR have circled around the development of new antimicrobials and the rationalisation of their use via various configurations of regulation of access and distribution, promotion of diagnostics and education of prescribers and consumers. Research by historians and social scientists (HSS) are increasingly demonstrating the various limitations and unintended consequences of many of these approaches, while also seeking to propose not only different ways to study AMR as a problem, but also address it. Part of this, involves serious engagement with microbiological insights (i.e. related to the microbiome) and methods to move beyond the impasses of outdated concepts (e.g. germ theory), methodological reductionism and disciplinary boundaries. Based on our empirical research on AMR and human microbiome science, we demonstrate how AMR is a transdisciplinary problem requiring contributions from HSS’s research and expertise in order to devise socially meaningful and microbiologically effective solutions. We have identified four areas where such contributions would be beneficial: (1) policies (e.g. AMR policies still assume germ theory and operate within silos); (2) AMR solutions are human centred (i.e. neglect of the microbiome and pay limited attention to other nonhumans) vs one health; (3) epidemiological variables and microbiological discourse (i.e. often employ outdated anthropological and philosophical concepts, such as westernised, modern, traditional); (4) Rhetorics and lexicon (i.e. can be morally and conceptually simplistic, like ‘war’, ‘sweets’, ‘good’/’bad’ bugs, ‘irrational’).
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Molecular identification of antibiotic resistance genes of bacteria isolates from ready to eat food and drinking water sold in Imo State University
More LessFood and water are fundamental need of human as its quality is of great concern to the generality of consumers, despite quality control measures during their production, unacceptable microbiological load often makes them unsafe. In this study, we analyzed various ready to eat food and water for presence of coliform bacteria, fungi count, antibiotic resistance gene and plasmid DNA, Samples were found to be positive for various antibiotic resistance genes namely, Sul1, Sul2, Tet A and TetB, interestingly, considerable total coliform bacteria were found from isolates and this result was further confirmed using illumina sequencing of the 16SrRNA gene. All Samples were negative for plasmid DNA, These finding deserve attention as the presence of coliform bacterial and antibiotic resistance genes potentiate health risk to members of university community and as such calls for strigent supervision and saftety and implementation of food safety regulations.
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Characterization of a multi-modular bacterial enzyme with binding and hydrolysis activities on fungal cell wall components: Insights into bacterial-fungal interactions in the soil
More LessBacteria from the phylum Bacteroidetes is known as good degraders because of their abilities in secreting glycoside hydrolases (GHs). As a member in Bacteroidetes, Chitinophaga pinensis is capable to hydrolyze several glycans (McKee et al., 2019). We found that a multi-modular CAZyme produced by C. pinensis contains not only two GHs but also two uncharacterized domains. Although it is common for CAZmes to have non-catalytic modules like carbohydrate-binding domains (CBMs), the domains in this protein are uncommon, and their functions unknown.
Our enzyme contains five domains – a GH5_46 enzyme, a GH18 enzyme, two non-identical uncharacterized domains, and a Por C-terminal secretion domain. I have shown that the GH5 can specifically degrade pustulan (β-1,6-glucan), and is the only β-1,6-glucanase found in its sub-family. The GH18 is a chitinase that reacts with β-chitin. Interestingly, the two uncharacterized domains both bind polysaccharides, and improve enzyme stability. However, they do not necessarily bind the same glycan as their GH partners. Our result demonstrates how two distinct polysaccharide-degrading modules within one protein can cooperate. We also discuss the impact of the binding domains on the GH activity.
We anticipate our research can stimulate the exploration of β-1,6-glucanase activity in family GH5, and expand the database of carbohydrate specificities in bacteria. The two unusual binding domains we have characterized suggest an unexplored aspect of bacterial physiology in the soil.
References:
McKee, L.S., et al., 2019. Focused metabolism of β-Glucans by the soil Bacteroidetes species Chitinophaga pinensis. Appl. Environ. Microbiol., 85(2), pp.e02231-18.
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Using structural biology of methanogen enzymes to aid our understanding of methane formation
More LessMethane is a potent greenhouse gas (28-fold more potent than carbon dioxide) and is a significant gas contributing to global climate change. Approximately a billion tons of methane are produced each year by methanogenic archaea in ruminants. These archaea possess a number of unusual traits such as isoprenoid-based lipids, unusual cell wall chemistry and a unique energy metabolism (methanogenesis) that requires six methanogen-specific cofactors. Many of the enzymes involved in these processes have no direct analogues in the host animal. To gain insights into the fundamental biology of rumen methanogens we have determined crystal structures of key enzymes with archaeal-specific traits. Over 600 enzymes were targeted for structure determination which produced approximately 200 purified soluble enzymes for crystallographic screening. More than 50 different enzymes have produced crystals and 30 structures have been solved for individual enzymes to date. The results have helped illuminate our understanding of methane formation at this critical juncture in the world’s history.
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Can m6A-modified RNA be used as an Anti-Viral Target?
More LessKaposi’s sarcoma-associated herpesvirus (KSHV) is a DNA virus associated with several HIV-associated malignancies.
Like all herpesviruses, KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND-1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019).
Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner. Taking this into consideration, novel ligands have been assessed as effective anti-viral reagents. The importance of A versus m6A within the lytic phase of KSHV’s lifecycle will be investigated by combining in silicoscreening with biophysical techniques and cell based assays.
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Identification of potential key genetic factors in the long-term success of Shigella as pathogens
More LessBacterial of the genus Shigella are a major contributor to the global diarrhoea burden causing 100,000 deaths per annum globally. Further to this, increasing antibiotic resistance in Shigella and the lack of a licenced vaccine has led WHO to recognise Shigella as a priority organism for the development of new antibiotics. Understanding what drives the long-term persistence and success of this pathogen will thus aid the global management of shigellosis and may identify targets relevant to other enteric bacteria. To identify key genetic drivers of Shigella evolution over the past 100 years, we have used the historical Murray collection; comprising several hundred pre-antibiotic era (1917 – 1954) Enterobacteriaceae from diverse geographical locations. We employed genome-wide association studies to sequences from over 100 Shigella isolates from the Murray collection alongsidemore modern (i.e. 1950s – 2018) isolates to identify genetic factors (SNPs and genes) significantly associated with time as a continuous variable. GWAS hits (e.g. a putative resistance protein) then underwent variation, functional and phenotypic analysis to examine the plausibility of their role in shaping Shigella populations and as potential targets for managing this pathogen.
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Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Despite on-going vaccination programmes, Neisseria meningitidis causes over 700 cases of invasive meningococcal disease (IMD) in the UK each year. In 2017-18, the MenW and MenY capsular groups caused 38% of all IMD cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to understand how genetic variation contributes to phenotypic differences between carriage and disease isolates.
We are adapting a variety of assays, designed to mimic carriage and disease behaviours, for high throughput phenotypic testing of multiple meningococcal isolates from carriage and cases of IMD. We have selected 335 MenW cc11 and MenY cc23 isolates and are currently testing subsets of isolates in cell culture (CaLu3), growth and biofilm assays. Phenotypic differences will be utilised as input data for Genome Wide Association Studies that aim to identify the specific genomic variants, or combinations of variants, determining observed differences. Genomic data will include whole genome sequences and repeat-mediated phase variation states.
Our preliminary data has detected variation in the ability of cc11 and cc23 isolates to disrupt monolayers of CaLu3 cells, indicating that minor genetic differences in phylogentically similar organisms may be physiologically important for both carriage and disease. We will also discuss progress in establishing successful, high-throughput assays for testing multiple isolates.
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Digital Outreach: Promoting a global understanding of antimicrobial resistance (AMR)
More LessTechnology allows educators to reach a global audience without amassing a large carbon footprint. The Skype in the Classroom programme from Microsoft enables me to spark curiosity and engage a wide range of students in dialogue to explore global issues, in particular, the threat of antimicrobial resistance, while supporting me to develop my skills as an educator. Through a multi-disciplinary approach, I employ Skype in the Classroom as a future-proof, powerful tool in our arsenal against the evolving microbial threat. We are not only responsible for teaching the next generation of scientists, but also the next generation of thought-leaders and professionals, who will work collaboratively towards our common goals.
Antimicrobial resistance is one of the grand challenges of the 21st century, and global connectivity is one of the best solutions to promoting better understanding of this threat. The Skype in the Classroom programme has allowed me to teach sessions to students in Norway, India, Brazil, Miami, Florida and Puerto Rico, with students ranging from 10 to 18 years old. I use skype and Teams to deliver online lessons in an accessible way, starting with the scope of the problem, covering the technical detail of how resistance emerges and is spread, and looking at some of the newest approaches to tackling AMR. Using a ‘challenge-based-learning’ approach, we discuss how different professionals and roles within society can contribute to the issue. This makes the class applicable to students with a wide range of backgrounds, including those without a keen interest in science.
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