Rotation Projects
ABC transporters and commitment to sexual development in malaria parasites
Supervisor: Prof. Andy Waters
Rotation project summary:
Plasmodium spp. parasites are responsible for malaria and cause significant mortality and morbidity on human populations with further consequent impact on the global economy. Transmission of malaria parasites results from sexual development that generates male and female sexual precursor forms (gametocytes) that are taken up as part of the blood meal by a female anopheline mosquito. Initiation of sexual development of malaria parasites in the blood is in part controlled by the environment. Through a systematic gene deletion approach an ABC class transporter that is predicted to be an antiporter has been identified that has significant impact on the ability of the rodent malaria parasite Plasmodium berghei, to commit to sexual development. Whilst there is a very small basal number of gametocytes produced no environmental perturbation has been identified that affects this level of commitment. Using comparative metabolomics studies, we will attempt to identify the substrate of this transporter as a prelude to the identification and mechanistic characterisation of a mechanism Plasmodium parasites use in order to gauge the optimal level of investment into gametocytogenesis. The outcomes of this research could lead to novel methods to control/prevent malaria parasite transmission.
References:
Kent et al (2018) Nature Microbiology PMID 30177743
Lee, R.S. Waters, A.P.* & Brewer J.M.* (2018) Nature Communications. PMID 29703959.
High-throughput cryo-electron microscopy of influenza haemagglutinin molecules as a tool to inform vaccine design and to study antigenic drift.
Supervisor: Prof. David Bhella
Project Title: Structure analysis of SARS-CoV2 spike protein maturation and evolution
Rotation project summary:
The spike protein (S) of SARS-CoV2 is the viral fusion protein that mediates attachment and entry to the new host cell. This large glycoprotein assembles as club-shaped trimers on the outside of the viral envelope. Like all viral fusion proteins, S is expressed as a single polypeptide chain that is post-translationally cleaved into its active state. We aim to understand how the protein has adapted to enter human cells, focussing on the role of host proteases in protein maturation.
Aim(s): In this project we will use cryo-EM to solve the structure of the S protein in complex with furin, tmprss2 and cathepsin the host proteases that regulate fusion activity by cleaving S protein from its precursor form to its active form. Adaptation of protease specificity has been a key driver of viral adaptation to the human respiratory tract. We will also investigate the impact on protein conformation of introducing mutations in the receptor binding motif and N-terminal domains that have been found to modulate entry pathways.
Training outcomes: The trainee will gain experience in cloning, protein expression and purification, transmission electron microscopy, cryo-EM, 3D image reconstruction and the construction of atomic models of protein structures.
References:
Cantoni, D. et al. (2022) Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike. EMBO Reports, (doi: 10.15252/embr.202154322) (PMID:35999696)
Willett, B. J. et al. (2022) SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway. Nature Microbiology, 7(8), pp. 1161-1179. (doi: 10.1038/s41564-022-01143-7) (PMID:35798890) (PMCID:PMC9352574)
Project Title: Structure and assembly of respiratory syncytial virus
Rotation Project Summary:
Respiratory syncytial virus is a leading cause of paediatric respiratory disease, causing bronchiolitis in neonates and infants. RSV is a negative-sense, single-stranded RNA containing enveloped virus and assembles filamentous virions at the host cell plasma membrane. We have recently published the intermediate resolution structure of the matrix protein (M) layer, which forms a helical lattice underneath the viral envelope and is critical in coordinating virion assembly. We also found that the viral glycoproteins (F and G) are helically ordered on the viral envelope, and that this is coordinated by the underlying M array.
Aim(s): In this project we will use cryogenic electron tomography to solve the high-resolution structure of the M-layer, with the aim of resolving the molecular contacts between M and F/G. We will also use tomography to image the site of virion assembly, to understand how alternate packing of M and F/G may define virion structure and assembly.
Training outcomes: The trainee will gain experience in virus culture, transmission electron microscopy, cryo-ET, 3D image reconstruction by sub-tomogram averaging and the construction of atomic models of protein structures.
References:
Conley, M. J. et al. (2022) Helical ordering of envelope associated proteins and glycoproteins in respiratory syncytial virus. EMBO Journal, 41(3), e109728. (doi: 10.15252/embj.2021109728) (PMID:34935163) (PMCID:PMC8804925)
RNA viruses, molecular evolution & recombination
Supervisor: Prof David L Robertson
Rotation project summary:
RNA viruses such as HIV-1, influenza and coronaviruses tolerate high levels of variation. This genetic change is generated during an infection as the virus replicates or by host anti-viral molecules such as APOBEC. Variation can contribute to new virus phenotypes, host-switching, immune and vaccine escape, or evasion of anti-viral host-molecules and drugs. While the rate of virus evolution is often extreme, this mostly does not necessarily lead to functional change. First, most tolerated mutations are ‘neutral’ either not changing the amino acid or resulting in conservative amino acid replacements. Second, viral genes code for amino acid sequences that must retain the ability to fold into functional protein structures. Third, the virus is heavily constrained to interact with the same host machinery. The aim of this computer-based project will be to use knowledge of functional constraints to investigate genotypic change in publicly available data for the chosen virus that is likely to have a phenotypic consequence. This will involve exploring the large sequence data sets available for studying viral variation. The rotation will focus on one of the viruses of most relevance to human health and study protein domain variability and conservation, linking these to virus-host interactions data sets.
Defining the genetic signature of malaria transmission
Supervisor: Prof. Matthias Marti
Rotation project summary:
Malaria is a global health threat with more than 200 million cases and 400’000 deaths each year, most of them cause by Plasmodium falciparum. The goal of this project entitled “Functional analysis of natural drivers for transmission in malaria” is to define and functionally validate genetic determinants of malaria transmission. We hypothesize that the parasite’s ability to adapt to short- and long-term variation in environmental conditions (between individual hosts, across geographic regions and species) is determined by a combination of genetic and epigenetic factors. Recent work by the PIs has identified genetic loci linked to transmission that are under strong diversifying selection in P. falciparum, and lineage-specific expansion or loss of other loci across the Plasmodium genus. In addition, the PIs have spearheaded development and implementation of single cell OMICs, and novel computational methods to process NGS data. Here we propose the following approaches to systematically mine available datasets: i) Identify signatures of diversifying selection driving variation in transmission rates across P. falciparum strains, and ii) identify lineage-specific changes over evolutionary scales related to transmission across Plasmodium lineages. Second, we will functionally investigate these loci using reverse genetics in P. falciparum and the rodent parasite P. berghei.
Co-PI: Dr. Thomas Otto.
References:
1. Brancucci NMB, et al. (2017) Cell 171:1.
2. Rutledge GG, et al. (2017) Nature 542(7639):101.
Identification of the molecular target of a species-specific antibiotic active against drug-resistant Pseudomonas aeruginosa
Supervisors: Prof. Daniel Walker
Rotation project summary:
The increasing prevalence of antibiotic resistant Gram-negative bacteria poses a catastrophic threat to the population and despite obvious clinical need few new antibiotics have entered clinical practice in recent years. The lack of novel classes of antibiotics in development is in part due to the relative failure of post-genomic target driven antibiotic discovery, which has yielded few novel antibiotic targets and consequently few candidate small molecule drugs for further development. In contrast, evolution has an excellent track record at identifying cellular components against which antibiotics can be targeted. This is reflected in the mostly naturally derived antibiotics currently used in clinical practice. We have identified a highly targeted protein antibiotic, pyocin SX2, with potent activity against Pseudomonas aeruginosa. Preliminary work has shown that this antibiotic kills cells through interference with RNA processing, suggesting a completely novel mode of action. In this project we will use a combination of biochemical, biophysical and transctiptomic experiments to identify the target of pyocin SX2 and uncover the molecular basis for its killing activity.
Impact of environmental change on African malaria vectors
Supervisor: Prof. Heather Ferguson
Rotation project summary:
In the last decade, substantial shifts in malaria vector species composition have been reported in several African settings, with the prolific vector Anopheles gambiae being replaced by the less efficient An. arabiensis. Another vector Anopheles funestus has remained present at relatively low densities, but is increasingly recognized as the major source of remaining transmission. These changes in vector species composition and their role in transmission have been attributed to their differential susceptibility to insecticides. However climate has also changed over the same period that interventions have been introduced across Africa, raising the possibility that long-term environmental change may also have an unacknowledged role. Using a variety of Anopheles colonies maintained at UoG, here you will conduct experiments to evaluate the differential of impact of environmental variation on several African malaria vector species. Skills acquired will be mosquito rearing and fitness measurements, experimental design, and quantitative analysis. Note should restrictions on laboratory work remain during the project period, a “dry” version of this project is possible in which long-term data sets on climate and vector species composition can be mined to carry out a meta-analysis of the impact of environmental change on different vector species.
Dissecting the divergent and essential mitochondrial complex II of malaria and Toxoplasma,
Supervisor: Dr Lilach Sheiner
Rotation project summary:
Apicomplexan parasites, including Plasmodium and Toxoplasma, cause diseases of global importance such as malaria and toxoplasmosis, for which drugs are still needed. The apicomplexan mitochondrion is important for parasite energy and metabolism, and is a proven target for antimalarial and anti-toxoplasmosis drugs (for example, Atovaquone). Recent discoveries suggest the apicomplexan mitochondrial complexes are highly divergent from their mammalian hosts (e.g. PMID: 33651838).
Despite their importance for parasite survival and the evidence of being different from the host and known targets for drugs, we still don’t know the composition and functional mechanism of mitochondrial complexes in apicomplexans. One barrier has been the lack of tools to isolate these complexes for proteomic and structural studies, partly since Plasmodium in vitro culture poses a challenge for biochemical studies that rely on high quantities of material. We had overcome this difficulty through using the related parasites, Toxoplasma, for which we developed a new mitochondrial isolation technique (PMID: 33402698) and via the application of advanced proteomics methods. This enabled us to identified novel potential complex components that are conserved among the two parasites and absent in the human and animal hosts.
In this rotation project, we will validate the new components through genetic tagging, high-end microscopy and through biochemical methods. This will establish the means to study how these complexes work and potentially how inhibitors might interfere with their function, which will lay the foundation for future drug development efforts.
In addition to learning those techniques, the student will be exposed to the various projects in the lab, addressing different aspects of the cell biology of both Plasmodium and Toxoplasma: https://lilachsheiner.wixsite.com/sheinerlab-wtcmp/current-lab-members; and will be encouraged to devise a plan for their own project going forward.
Paper that represent other ongoing projects in the lab:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851545/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823475/
https://www.biorxiv.org/content/10.1101/481192v1
C. difficile and inflammation: what role does the S layer play in immune cells signalling?
Supervisor: Dr Gillian Douce
Rotation project summary:
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea worldwide and continues to cause significant morbidity and mortality, with a consequent healthcare cost burden of over €3B in the EU and $4.8B in USA (1). In severe CDI, hyper-induction of inflammatory processes and massive infiltration of neutrophils are linked to significant complications and poor patient outcome.
Unusually for a human pathogen, C. difficile is encased in a semi-rigid proteinaceous coat, known as the S-layer, which is largely composed of a single protein SlpA. This structure has been linked to several key pathogenicity traits including epithelial adhesion and activation of the immune response through TLR4 signalling (2). In vivo, modification of the S layer results in reduced disease (weight loss) and limited inflammatory influx and epithelial damage.
This project will determine whether reduction of inflammatory influx is a consequence of reduced TLR signalling. This will require the testing of genetically modified strains of the organism that express structural varients of SlpA on TLR-2, TLR-4 and TLR-5 reporter cell lines. Further, there is scope in the project to determine if there is any synergistic link between SlpA, toxin secretion and inflammation through the use of toxin producing and atoxic strains.
- Ryan A, Lynch M, Smith SM, Amu S, Nel HJ, McCoy CE, Dowling JK, Draper E, O'Reilly V, McCarthy C, et al. A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins. PLoS Pathog 7:e1002076.J Med 372:825-834.
- Kirk, J. A., Gebhart, D., Buckley, A. M., Lok, S., Scholl, D., Douce, G. R., Govoni, G. R. and Fagan, R. P. (2017) New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. Trans. Med, 9:406 eaah6813.
Understanding cell fate decisions of anti-viral cytokine-producing memory T cells
Supervisor: Dr Megan MacLeod
Rotation project summary:
Influenza A viruses (IAV) cause significant morbidity and mortality through annual epidemics and pandemics every 50-100 years. Current vaccines generate antibodies that only protect against strains with viral surface proteins that match the vaccine strain. IAV specific T cells, in contrast, can recognise conserved viral proteins providing cross-strain immunity. Our ability to design vaccines that generate long-lived protective T cells is limited by our inadequate understanding of the activation signals and environmental cues required to generate these cells.
We have demonstrated that IAV specific memory CD4 and CD8 T cells change as they develop and survive as memory cells. These cells increase their capacity to produce multiple cytokines. We aim to investigate what regulates this improved cytokine production in memory T cells to understand how vaccines could improve the generation of long-lived protective T cells.
In this project, we will examine the expression of a number of different cell surface and intracellular molecules by IAV specific cytokine producing T cells at different times following infection. This will provide key insights into the cell fate decisions that determine why multiple-cytokine producing T cells come to dominate the memory pool. This project will focus on cell culture and multi-colour flow cytometry techniques.
Understanding how DNA replication programming drives adaptive genome variation in African trypanosomes and Leishmania
Supervisor: Prof. Richard McCulloch
Rotation project summary:
DNA replication is a central reaction in life, providing for genome inheritance and maintenance of cell function during growth and development. To ensure accurate genome transmission, eukaryotic cells have evolved tightly controlled programmes of DNA replication in which new copies of the genome are generated from multiple loci termed origins, where DNA synthesis initiates during S-phase of the cell cycle. Origins are defined by binding of an initiator complex, termed the Origin Recognition Complex (ORC), leading to a cascade of replication factor recruitment and activation. Despite considerable mechanistic understanding of these events, many questions remain: what features dictate origin location and function; how flexible is the origin-driven programme of DNA replication; and when can cells employ origin-independent DNA replication? Answering these questions is critical to understand how genome content evolves, either incrementally during evolution, or abruptly to adapt cell behaviour in response to environmental change.
An understanding of eukaryotic DNA replication is largely derived from work in a limited number of organisms, including yeast, mammals and Drosophila, and findings have been generalised across this diverse domain of life. This application seeks to build upon work in two important eukaryotic parasites, Trypanosoma brucei and Leishmania, which has suggested divergence in the DNA replication programme and machinery relative to other characterised eukaryotes. A major potential deviation from other eukaryotes derives from whole genome mapping analyses, which suggest these parasites replicate their genomes with insufficient numbers of ORC-defined origins to complete replication of their complete genomes during S-phase. If so, such origin paucity may indicate that DNA replication flexibility in central to the biology of these parasites, perhaps allowing elevated rates of genome sequence change. Such sequence change may be crucial for survival and transmission, such as by antigenic variation.
This project will seek to test the predicted organisation of DNA replication in the two parasites. To do so, next generation sequencing strategies, some using long-read Nanopore approaches, will be deployed to detect and characterise all locations of DNA replication in single cells, as well as to look at patterns of DNA synthesis, including rate, pausing and termination. In addition, the project will engineer the genomes of the two parasites using CRISPR and site-specific recombination to change the number of origins, as well as move origins from known locations into putative origin-free genome regions, thereby testing the constraints of DNA replication programming.
The project will involve molecular and cellular biology, genetic modification and culture of parasites, and bioinformatics. A recent review of this topic from our lab is below:
Damasceno, J. D., et al. (2021). "Read, Write, Adapt: Challenges and Opportunities during Kinetoplastid Genome Replication." Trends Genet 37(1): 21-34.
New threats from old friends
Rotation project summary:
This work builds on our many years of work on E. coli, a model organism that is both a pathogen and workhorse of molecular biology. In this work we are interested in the evolution and emergence of new hybrid strains of E. coli. We have been collaborating with a group in Brazil that identified novel strains that do not fit the normal classification we use for E. coli. They contain genes normally seen in gut pathogens but also many of the genes seen in bladder specific pathogens. The strains have been sent for sequencing with the data available by the start of this project. For the lab component, we would see which virulence proteins the bacteria can express and secrete and look at how the strains interact with host cells. Typically strains carry around 30-50 secreted proteins but we have no idea for these novel strains. The work would involve some bioinformatics, molecular biology and even proteomics. It's an interesting way to explore new strains that are involved in disease. The project has enormous scope and fantastic tools to explore these novel isolates.
A good paper to look at would be:
Draft Whole-Genome Sequence of a Uropathogenic Escherichia coli Strain Carrying the eae GeneMicrobiology Resource Announcements
2019-10-24 | journal-article, DOI: 10.1128/MRA.00980-19
Elevational gradients in infection and immunity in vampire bats
Supervisor: Dr. Daniel Streicker
Rotation project summary:
Adaptations to deal with challenging environmental conditions in high elevations may create trade-offs with pathogen defence. Such trade-offs may be most pronounced in non-humans given the absence of technological and medical interventions, but few taxa sufficiently broad elevational gradients for investigation. Our lab studies populations of vampire bats in Peru that range from sea level to over 3500 meters. Interestingly, genetic data suggest that bats invaded the Andes from the Amazon multiple times (Streicker et al. 2016 PNAS). This creates a naturally-replicated experiment in high elevation adaptation in a species with extreme metabolic challenges arising from flight and a diet of blood. Our studies also revealed elevational gradients in viral diversity (Bergner et al. 2020 Molecular Ecology) and altered immunity at geographic range limits (Becker et al. 2019 Int. Comp. Bio.). This project aims to establish a vampire bats model for relationships between elevation, infection and immunity. Depending on lab accessibility and student experience, the rotation will either generate genomic sequences from bats across Peru, mine existing metagenomic data, or have a literature review focus. Initial objectives include characterization of the number and timescale of Andean invasions or identification of regions of the bat genome under environmental or pathogen-mediated selection.
Identifying the antiviral defences that constrain the emergence of coronaviruses in human populations
Supervisor: Dr Sam Wilson
Rotation project summary:
Viruses continue to emerge and re-emerge in human populations with alarming frequency. The COVID-19 pandemic began when SARS-CoV-2 was transmitted from horseshoe bats to humans (possibly via an intermediate host such as pangolins). Every species posses its own repertoire of antiviral defences (Shaw et al., 2017), and this repertoire in part determines which viruses can successfully emerge in that species. Our group is interested in how genome encoded antiviral defences inhibit virus transmission and constrain viral emergence in human populations (Rihn et al., 2019).
The major aim of our laboratory is to understand which antiviral defences can target viruses and why these defences sometimes fail to prevent cross-species transmission (which can result in major pandemics such as recent influenza A, HIV-1 and COVID-19 pandemics). This rotation project is tailored towards SARS-CoV-2/COVID-19 but could equally be applied to other viruses (such as influenza A viruses or ebola virus, depending on the interests of the student and our reagent base/biosafety infrastructure).
AIM1 – To complete biosafety training and learn to work at containment level 3 (CL-3) in the Richard Elliott Biosafety Laboratory (REBL). SARS-CoV-2 must be handled at CL-3, so it is important to commence training at the earliest opportunity. This typically takes 1-2 months, with the majority of this time spent completing experiments alongside a buddy.
AIM2 - We have recently completed a large screen aimed at identifying antiviral interferon stimulated genes (ISGs) that inhibit SARS-CoV-2. The project will involve making cell lines that exogenously express these defences and examining their effect on SARS-CoV-2.
AIM3 – Whilst access to the REBL is limited (due to training), the student will examine the ability of the same factors to inhibit seasonal CoVs (CL-2).
Likely techniques: Cell culture, virus propagation, plaque assay, flow cytometry, high content imaging, western blotting, molecular cloning, lentiviral vector production, generation of modified cell lines
Rihn, S.J., Aziz, M.A., Stewart, D.G., Hughes, J., Turnbull, M.L., Varela, M., Sugrue, E., Herd, C.S., Stanifer, M., Sinkins, S.P., et al. (2019). TRIM69 Inhibits Vesicular Stomatitis Indiana Virus. J Virol 93.
Shaw, A.E., Hughes, J., Gu, Q., Behdenna, A., Singer, J.B., Dennis, T., Orton, R.J., Varela, M., Gifford, R.J., Wilson, S.J., et al. (2017). Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses. Plos Biol 15.
Understanding host-parasite interactions using single cell transcriptomic analysis of trypanosome parasites and their mammalian host.
Supervisor: Prof. Annette MacLeod
Rotation project summary:
Human African Trypanosomiasis (HAT) is a devastating neglected tropical disease affecting vast regions in rural Africa. It is caused by the protozoan parasite, Trypanosoma brucei gambiense and transmitted by tsetse flies. One of the hallmarks of HAT is the low number of parasites found in blood, however, recently we have discovered that a significant number of trypanosome parasites also reside in the skin of their hosts. These skin-dwelling parasites are particularly important as they contribute to disease transmission when the tsetse vector takes a bloodmeal.
The central aims of this project are:
1) to understand how trypanosomes colonise, functionally adapt and thrive in host’s skin and
2) to understand the intrinsic responses of the human skin to T. b. gambiense infection.
To achieve this, we will employ dual host-pathogen single-cell transcriptomics analysis using mouse models of infection and from skin biopsies derived from HAT patients.
We anticipate that the outcome of this project will inform about parasite and host genes that are up or down-regulated upon skin invasion, thus enabling us to generate further hypotheses-driven research questions that will be built upon these datasets.
Iron and immunity in Toxoplasma gondii infection
Rotation project summary:
Iron is essential for almost all life. As a key component of enzymes required for DNA replication and energy production, organisms must acquire and store iron from their environments. One of the major uses of iron is in the immune system, where simultaneously iron is sequestered to prevent access by pathogens, and is required for immune cell proliferation.
For intracellular parasites, separated from their host’s bloodstream, iron acquisition presents extra challenges. Toxoplasma gondii is an obligate intracellular parasite, and little is known about how Toxoplasma subverts its host to acquire essential nutrients like iron. Toxoplasma infects approximately 30% of people worldwide, and can cause severe disease. Understanding how iron acquisition regulates parasite survival and host pathology could reveal new therapeutic targets.
To interrogate how Toxoplasma acquires iron from its host cell, and how Toxoplasma growth is affected by host iron metabolism, we will use several exciting techniques established in the lab, including CRISPR/Cas9 genetic manipulation, multi-well growth assays, microscopy, cell culture and flow cytometry. We will also assess how Toxoplasma’s subversion of iron affects the survival and function of the host cell. Toxoplasma can infect every cell type, but particularly targets immune cells, using them as trojan horses to disseminate throughout the host. Immune cells are highly dependent on access to iron, and we will also investigate how parasite regulation of iron metabolism can mediate the contest between effective immunity and parasite evasion.
Identification of new and emerging viral genomes in Uganda
Rotation project summary:
The ArboViral Infection study in Uganda is a project designed to identify known and unknown viral pathogens in samples derived from patients presenting to hospital with fever and in the surrounding environment. Samples obtained from rodents, mosquitoes, bats and ticks have been obtained and subjected to next generation sequencing.
https://www.gla.ac.uk/researchinstitutes/iii/cvr/clinicalresearch/
Rotation projects are available to carry out next generation sequencing bioinformatic analysis and to develop serological assays for pathogens found as part of the study.
Molecular epidemiology of Coxiella burnetti
Rotation project summary:
Coxiella burnetii is a bacterial pathogen that causes febrile disease in people and significant production losses in economically crucial livestock species (e.g. cattle, sheep and goats) globally. C. burnetii is endemic in Tanzania, where C. burnetii infection, also known as Q fever, was diagnosed in 5.0% of people with acute febrile illness (Prabhu et al. 2011). Serological testing reveals that C. burnetii exposure is common in livestock and bacterial carriage and shedding was detected in 17% of goats,4% of cattle and 2% of wild rodents [Halliday et al. in prep], providing evidence of the complex multi-host epidemiology of this important pathogen in Tanzania. There is evidence that C. burnetii genotypes vary in their pathogenicity and epidemiology, but data about the distribution and clinical significance of these genotypes in endemic settings like Tanzania is lacking. This project focuses on the generation and analysis of molecular data to advance our understanding the epidemiology of C. burnetii in Tanzania. For a mini-project the student would work to analyse existing multi spacer typing and sequence capture data to characterise the diversity of Coxiella genotypes present in people, livestock and wildlife samples from Tanzania, with scope for additional laboratory data generation.
Drosophila as a model to study local and systemic impact of intestinal pathogenic infection.
Rotation project summary:
The adult intestine is a major organ with vital physiological, endocrine, immune and metabolic roles. These functions are achieved by stem cells and specialized cells such as absorptive enterocytes and hormone-producing enteroendocrine cells (EEs). Using Drosophila melanogaster, we have recently identified novel roles of EEs in intestinal and whole-body homeostasis (1, 2). Interestingly, intestinal infection with pathogenic bacteria leads to very significant changes in EEs in Drosophila and mammals. We hypothesize that intestinal pathogenic infection has significant systemic impacts, which are mediated by EEs.
This mini project will involve the use Drosophila to:
- Characterize enteroendocrine hormone changes in the intestine following pathogenic infection.
- Studying the impact of intestinal pathogenic infection in systemic functions such as feeding, sleeping and metabolic homeostasis.
1- Scopelliti A*, Cordero JB*1, Diao F, Strathdee K, White BH, Sansom OJ, Vidal M1. Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut. Curr Biol. 2014 Jun 2;24(11):1199-211. *Co-First; 1Co-Corresponding
2-Scopelliti A1*, Bauer C*, Yu Y, Zhang T, Krüspig B, Murphy DJ, Vidal M, Maddocks OK, Cordero JB1. A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila. Cell Metab. Oct 15. pii: S1550-4131(18)30629-6. *Co-First; 1Co-Corresponding
How malaria parasite gene expression adapts to mosquito host: in-depth analysis of Plasmodium berghei transcriptome during the initial stages of transmission
Rotation project summary:
To pass from one mammalian host to another the malaria parasite needs to be transmitted by an insect vector. During the initial stages of this process the specialised parasite stages (gametocytes) are ingested by a mosquito and transform into motile invasive forms (ookinetes) able to establish midgut infection. This transition is the biggest bottleneck of the parasite life cycle and, in theory, a great target for transmission-blocking interventions. It is however largely understudied.
The aim of this project is to generate gene expression profiles of the developing P.berghei ookinetes isolated from mosquito midguts at different time points. This data will be compared with similar datasets from parasites differentiated in vitro in order to map the similarities and differences between the two populations, and consequently to establish the limitations of in vivo and in vitro models. The project will familiarize the student with the Plasmodium transmission through mosquito (including isolation and quantification of different parasite stages) and generation of ATAC-seq and RNA-seq NGS libraries (both bulk and single-cell ones) from the parasite material.
In case the wet-lab projects are impossible, the student will focus and on the analysis of recently generated bulk and single-cell RNA-seq datasets from in vitro differentiated ookinetes.
Investigating the interactions of a novel tick-borne Banyangvirus with the mammalian innate immune system.
Rotation project summary:
Zweisel bat banyangvirus (ZbbV) is a virus that has recently been discovered in Germany, isolated from dead Eptesicus nilssonii (Northern bats). A recent study has shown that it is classified within the Banyangvirus genus of the Phenuiviridae family of viruses (1). Due to close phylogenetic clustering of ZbbV with other known banyangviruses such as severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) it is believed that ZbbV will be a tick-borne pathogen. No data is available on the ability of ZbbV to cause human disease and there are no serological data available to show where ZbbV is endemic.
Previous work in the laboratory confirmed multiple interactions between the non-structural (NSs) proteins of HRTV and SFTSV with mammalian innate immune components such as TBK-1, STAT2 and IRF-3. These viruses are severe human pathogens with a high case fatality rate. While at the same time, we showed that the NSs of an apathogenic virus (Uukuniemi virus; UUKV), was only able to weakly interact with MAVS (2).
This project will utilise protein expression systems for the NSs of ZbbV to examine its potential in antagonising the mammalian innate immune system and confirm its status a likely human pathogen.
References
- C. Kohl et al., Zwiesel bat banyangvirus, a potentially zoonotic Huaiyangshan banyangvirus (Formerly known as SFTS)-like banyangvirus in Northern bats from Germany. Sci Rep 10, 1370-1370 (2020).
- V. V. Rezelj et al., Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins. mSphere 2 (2017).
Integrating viral genome sequences and host receptor structure to predict the host range and tissue tropism of RNA and DNA viruses with machine learning
Rotation project summary:
We have recently shown that single stranded RNA viruses (e.g. ebolavirus, coronavirus, zikavirus) encode in their genomes signals that point to the taxonomic identity of their reservoir hosts, whether they are transmitted by a vector and if so, the taxonomic identify of that vector using machine learning to analyse the genomic sequence of ~500 viruses (Babayan, Orton, Streicker, 2018 Science). Analogous models may be useful to predict the host range of viruses more broadly (i.e., the other species which might be infected in the future) but require additional sources of information to make the most realistic actionable predictions. The ability of viruses to bind host receptors is a key prerequisite for infection, and as recently shown in the context of SARS-CoV-2’s spike protein, viruses hosts can become ‘pre-adapted’ to the receptors of other related species while still circulating in their natural reservoirs (MacLean et al. 2020, bioRxiv). The expression of appropriate receptors across tissues may further predict the tissue tropism and degree of pathogenicity following infection. Therefore, we predict that combining the genomic signals that underlie the ability of our models to accurately predict reservoir hosts with information on receptor binding could produce models that accurately predict the host range and pathogenicity of diverse viruses.
In this project, we will therefore extend our approach to new virus groups including double-stranded RNA such as bluetongue virus and DNA viruses such as herpes simplex virus and varicella zoster virus. This will form the core of the rotation project.
We will next seek to incorporate structural features of both virus and host receptors, e.g. the secondary protein structure of host receptors, into our models to enrich their predictive capacity and increase our ability to infer both the range of host taxa that viruses could infect and the potential effects of their infection on the organism. Finally, this project will also offer the opportunity to validate model predictions in vitro. The successful candidate will work under the supervision of Simon Babayan and Daniel Streicker, and in close collaboration with the Institute of Biodiversity, Animal Health & Comparative Medicine, and with the MRC-University of Glasgow Centre for Virus Research.
High-dimensional investigation of the blood-brain barrier in cerebral malaria.
Rotation project summary:
Cerebral malaria is the most serious and deadly complication of malaria. Cerebral kills by causing severe brain swelling resulting from fluid leak into the brain: blood-brain barrier breakdown (BBB). A main focus of our lab is investigating the causes of BBB breakdown in order to identify treatment targets.
We are using multiple high-dimensional and Omics methods to understand the mechanisms behind BBB breakdown in both human samples in Malawi (Africa) and in a culture based BBB model in Glasgow. In Glasgow we also have a large biobank of postmortem tissue from fatal cerebral malaria cases to enable various advanced imaging modalities to examine the blood vessels and host:parasite interactions in the brain.
Plasmodium falciparum infected red blood cells (iRBC) sequester and stick in blood vessels in the brain. We hypothesise that BBB breakdown is caused by different parasite factors released from iRBC that cause changes to brain endothelial cells that lead to loss of tight junctions. High dimensional technologies offer the opportunity to systematically examine this and to link – by profiling – findings in postmortem samples and in vitro experiments.
The overall PhD project will focus around investigating the mechanism by which different factors released from iRBC cause BBB breakdown and will involve the option to do some of the work on patient samples in Malawi as well as significant mechanistic work using the BBB model in Glasgow. Potential techniques involved include single cell RNA-sequencing, Imaging Mass Cytometry, Confocal microscopy, high content imaging, cell and parasite culture and learning to analyse these different generated data.
The 10 week project will focus on the in vitro model and investigating changes to the transcriptome and phenotype of brain endothelial cells in response to different parasite factors using Next-generation sequencing and/or high content imaging. Identified candidates can be validated in postmortem tissue using fluorescence staining and confocal microscopy. The scope of the project is flexible and will depend on a candidates existing skills and priorities – but could include parasite and tissue culture, flow cytometry, high content imaging, Next-generation sequencing, immunofluorescence and confocal microscopy.
Given restrictions due to COVID-19 significant sequencing data generated in our lab offers an opportunity to start with analysing these data to identify candidates – which could then be followed up either in vitro or by using confocal imaging in tissue.
Predicting the molecular targets of ivermectin in human and veterinary helminths
Rotation project summary:
Parasitic helminths cause debilitating disease in human and animals. A small number of anthelmintic drugs are available to treat and limit infection, but widespread drug resistance threatens sustainable control [1]. To preserve anthelmintic efficacy there is a desperate need to better understand drug mode of action and the genetic mechanisms that confer resistance.
Ivermectin is an essential drug for human and animal health. Despite widespread use in people and animals since the 1980s, the precise mode of action remains unclear. Ivermectin is primarily a glutamate-gated chloride ion channel agonist, but also affects a wider range of ion channels in a concentration dependent manner [2]. Ivermectin inhibits helminth feeding, motility and reproduction but the phenotypic effect is species and life-stage dependent e.g. death of adult gastrointestinal worms versus sterilisation of adult filarial worms. There are also significant differences in drug sensitivity in closely related helminths, such as a 40 - 50x difference in sensitivity to ivermectin in different species of human hookworm [3].
These differences in phenotypic effect are proposed to reflect differences in the composition and expression of ion channels in different helminth species and life-stages. This project will examine genomic and transcriptomic datasets for parasitic helminths of medical and veterinary importance to investigate differences in sequence and expression of glutamate gated chloride channel subunits to identify the likely targets of ivermectin. This will provide the student with training in comparative genomics, transcriptomics and protein homology modelling. In light of Covid-19, this can be a fully ‘dry lab’ project but target validation will be undertaken in the molecular biology lab if appropriate.
References:
- Kaplan RM, Vidyashankar AN: An inconvenient truth: global worming and anthelmintic resistance. Vet Parasitol 2012, 186:70-78.
- Laing R, Gillan V, Devaney E: Ivermectin - Old Drug, New Tricks? Trends Parasitol 2017, 33:463-472.
- Richards JC, Behnke JM, Duce IR: In vitro studies on the relative sensitivity to ivermectin of Necator americanus and Ancylostoma ceylanicum. Int J Parasitol 1995, 25:1185-1191.
Validating protein kinases as a target for next generation anti-malarial drugs
Rotation project summary:
Our laboratory are leaders in understanding the basic biology and therapeutic value of malaria protein kinases. Using molecular parasitology methods and proteomic approaches this project will investigate fundamental aspects of essential Plasmodium falciparum protein kinases and validate these kinases as targets for drug development. Working closely with the malaria drug discovery community we will not only offer training in dissecting the biology of the malaria parasite but also in the investigation of the suitability of targets for drug discovery. This PhD is built from the successful validation of the malaria protein kinase PfCLK3 (Alam et al. Science (2019) 365, eaau1682) which we determined not only to be essential for the correct processing of RNA but also when inhibited rapidly killed the parasite at multiple stages in a manner that indicated the potential of PfCLK3 as a therapeutic target. Using PfCLK3 as our exemplar we are now investigating the potential of other protein kinase targets which emerged from our global study of essential malaria protein kinases (Solyakov et al. Nature communications (2011) 2, 565).
Ticks as vectors of Crimean-Congo Haemorrhagic Fever virus in Tanzania
Professor Sarah Cleaveland
Rotation Project Summary: Ticks as vectors of Crimean-Congo Haemorrhagic Fever virus in Tanzania
Crimean Congo Haemorrhagic fever is a zoonotic disease caused by a tick-borne orthonairovirus that can cause severe haemorrhagic fever in humans. The virus is known to circulate in livestock and people in Tanzania, but very little is known about the impact, epidemiology or ecology of the disease in Africa. This study will involve PCR analysis of nucleic acid extracted from ticks collected from livestock (cattle, sheep and goats) in Tanzania as part of cross-sectional studies of zoonotic pathogens in communities where people live in very close proximity to animals. Data will be analysed to investigate CCHFV infection prevalence in different tick species, and how this varies with livestock and human infection prevalence in different agro-ecological settings.
Effect of improved water access on Schistosoma mansoni transmission
Supervisor: Dr Poppy Lamberton
Rotation Project Summary:
Schistosomiasis is a neglected tropical disease infecting over 240 million people, mainly in sub-Saharan Africa. Transmission is intrinsically linked to poverty, driven by poor water, sanitation and hygiene (WASH) conditions. Individuals acquire infections when they contact fresh water containing cercariae, the infective larval stage of the parasite. Water contact occurs through behaviours such as water collection and bathing. Water contamination, and hence disease transmission through water contact, is high in areas with inadequate sanitation. Presently, mass drug administration is the main form of control, but improved WASH facilities are required to interrupt transmission and reduce reinfection. This PhD will address questions surrounding the effectiveness, and uptake, of a low-cost water biofiltration system to safely extract and treat lake water by passage through sand filters to make it safe for drinking, bathing and other domestic uses. The project will involve working in the laboratory in the UK and with Ugandan school children to assess reinfection levels in communities with and without access to treated water.
Specific objectives may include:
- Testing the efficacy of up- and down-flow sand filters in removing, and/or inactivating, schistosome cercariae in the laboratory.
- Working alongside highly trained field teams to assist community building of low-cost biofilters using locally sourced materials.
- Monitor mansoni infection and reinfection levels in school-aged children between communities with safe water pumps and those with no additional interventions.
Specific objectives may vary depending on the candidate’s preferences and Covid-19 restrictions, with scope for project development in multiple directions. The PhD will be based within Poppy Lamberton’s interdisciplinary group aimed at reducing community transmission of Schistosoma mansoni in Uganda and co-supervised by Stephanie Connelly, an engineer with extensive experience in implementing locally appropriate water treatment solutions. Skills can be gained in parasitology, epidemiology, engineering and social sciences depending on the student’s scope.