Integrating viral genome sequences and host receptor structure to predict the host range and tissue tropism of RNA and DNA viruses with machine learning
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-throughput cryo-electron microscopy of influenza haemagglutinin molecules as a tool to inform vaccine design and to study antigenic drift.
Influenza A virus undergoes antigenic drift in the face of population immunity. Consequently, vaccine efficacy wanes over time requiring new vaccines to be formulated every year. Decisions about vaccine formulation must be made many months before the onset of flu season to allow manufacturers to scale up production. It is important for manufacturers to understand the potential impact on antigenicity of mutations that emerge during this production window. Moreover, a better understanding of the role of changes in antigenicity wrought by mutations in each specific amino acid residue in the HA sequence would lead to better models of influenza virus evolution. Such models could eventually feed into vaccine formulation decision making.
While the protein databank has an abundance of HA structures, there is limited diversity in sequences solved. We aim to systematically sample the evolutionary history of HA and use high-throughput cryo-EM to solve the structures of a panel of HA molecules specifically chosen to address the diversity of this molecule across time and populations. As such this project encompasses fundamental structural biology research, virus evolution and aims to inform policy on the global scale.
Uncovering the roles of cellular RNA-binding proteins as master regulators of virus infection
Dr Alfredo Castello
RNA is a central molecule in the life cycle of RNA viruses, acting not only as mRNA, but also as a genome. It is thus unsurprising that viral RNA becomes a hub for critical host-virus interactions in infected cells. However, the complement of cellular RNA-binding proteins that interact with viral RNA has remained largely unknown. In the last years, the Castello lab have made important advances towards understanding the composition of viral ribonucleoproteins, developing cutting edge proteome-wide approaches to study protein-RNA interactions in infected cells. Applied to several viruses, including HIV-1 and SARS-CoV-2, these studies revealed a new universe of host-virus interactions with critical roles promoting or restricting infection. These new host-virus interactions thus hold great potential for the development of novel broad-spectrum antiviral therapies.
The PhD projects in the Castello lab will build on these important discoveries, aiming to answer the following fundamental questions:
- How do cellular RNA-binding proteins recognise viral RNA?
- What are the molecular mechanisms by which these critical host-virus interactions control the viral life cycle?
- Can these cellular proteins be exploited as targets for antiviral therapies?
To answer these questions, we will apply multidisciplinary approaches, spanning cellular and molecular biology, RNA biology, virology and computational sciences.
- Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection. Kamel, W., Noerenberg, M., Cerikan, B., […], Bartenschlager R., Mohammed, S., Castello, A.* Mol Cell. 2020. https://doi.org/10.1016/j.molcel.2021.05.023
- Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture. Perez-Perri, J.I., Noerenberg, M., […], S., Hentze, M.W. and Castello, A.*. Nature Prot. 2021 Jan;16(1):27-60
- System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection. Garcia-Moreno M, Noerenberg M, […], Mohammed S and Castello A*. Mol Cell. 2019 Feb 21. pii: S1097-2765(19)30037-1. doi: https://doi.org/10.1016/j.molcel.2019.01.017
Ticks as vectors of Crimean-Congo Haemorrhagic Fever virus in Tanzania
Rotation project summary:
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.
C. difficile and inflammation: what role does the S layer play in immune cells signalling?
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.
Impact of environmental change on Africa malaria vector communtii
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.
The link between infection and autoimmunity
It remains controversial whether infections such as malaria are immunosuppressive and reduce the prevalence of autoimmune disease in endemic countries.
Conversely, it may be that the immune/inflammatory activation caused by infection may be protective for those diseases but predispose to autoimmunity subsequently.
We therefore propose to test the hypothesis that ‘propensity to produce low affinity, promiscuous binding autoantibodies is protective against the early childhood inflammatory consequences of malaria infection but predisposes to autoimmunity in later life’.
To do this we will combine laboratory based model studies with assessment of relevant clinical cohorts to examine the potential links between infection and autoimmunity and investigate the mechanistic basis of this.
This will be done with partners in Glasgow, Malawi and Kenya.
- The role of infections in the emergence of non-communicable diseases (NCDs): Compelling needs for novel strategies in the developing world. Ogoina D, Onyemelukwe GC. J Infect Public Health. 2009;2(1):14-29. doi: 10.1016/j.jiph.2009.02.001. Epub 2009 Mar 5. PMID:20701857
- Cellular imaging in rheumatic diseases. Benson RA, McInnes IB, Brewer JM, Garside P. Nat Rev Rheumatol. 2015 Jun;11(6):357-67. doi: 10.1038/nrrheum.2015.34. Epub 2015 Mar 24. PMID: 25800215
Using CRISPR-Cas9 mutant screens to study Leishmania gene function
Leishmania are unicellular parasites that are transmitted by sand flies and cause disease in humans and animals. In the mammalian host, Leishmania are taken up by phagocytic cells of the immune system and then replicate intracellularly in the phagolysosome of macrophages.
How does this parasite survive inside macrophages, the very cells designed to kill invading microbes?
To answer this question, we developed a rapid high-throughput method for CRISPR-Cas9 gene editing in Leishmania and rapid phenotyping of barcoded mutants. These methods allow us to dissect the role of parasite genes in infectivity and life cycle progression.
We currently focus on the function of the parasite’s flagellum, which is required for swimming and attachment in the sand fly. During macrophage infection, the flagellum is remodelled to a structure resembling a sensory cilium, which may be used for host-parasite communication.
Through the study of mutant phenotypes we aim to discover how this sensory-type cilium helps infection of macrophages and parasite survival. This project offers the opportunity to acquire skills in cutting edge CRISPR-Cas9 genome editing methods and bioimaging.
Beneke T, Demay F, Hookway E, Ashman N, Jeffery H, Smith J, Valli J, Becvar T, Myskova J, Lestinova T, Sadlova J, Volf P, Wheeler RJ and Gluenz E (2019). Genetic dissection of the Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. PLoS Pathogens 15:e1007828.
Beneke, T., Madden, R., Makin, L., Valli, J., Sunter, J. and E. Gluenz (2017). A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. Royal Society Open Science 4(5):170095.
Molecular epidemiology of Coxiella burnetti
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.
Virulence-transmission tradeoffs and evolutionary implications for the rabies virus
The rabies virus is almost universally fatal, yet has an apparently low rate of transmission.
The basic reproductive ratio for rabies has been estimated to be very close to one across a range of settings (Hampson et al. 2009).
Although evolutionary theory hypothesizes that intermediate virulence maximizes pathogen fitness due to tradeoffs between virulence and transmission, there is limited empirical evidence to support this assertion (Blanquart et al. 20019).
Using data from detailed contact tracing of natural rabies infections we propose to investigate the extent to which variation in transmission and virulence is heritable.
We observe considerable variation in the incubation period and duration of infection, as well as the biting behavior and movement of rabid dogs and our preliminary evidence suggests correlated variability in these traits.
We propose to use these data to develop an epidemiological-evolutionary model to predict how correlations affect viral persistence at the population level.
Through individual-based simulations we will examine heritability in traits needed to maintain the observed variation and evidence of virulence-transmission tradeoffs during epidemic growth and decline.
Deep sequencing of rabies viruses from sampled individuals where the transmission relationship is known, and from different anatomical sites from the same individual will provide further opportunities to link phenotype with genotype and quantify within host viral diversity and transmission.Overall this project should lead to a better understanding of the process of viral infection, transmission and persistence and we expect will generate testable hypotheses to be explored with both epidemiological and genetic data.
- Hampson, K. et al. Transmission Dynamics and Prospects for the Elimination of Canine Rabies. Plos Biology 7, e1000053, doi:10.1371/journal.pbio.1000053 (2009).
- Blanquart, F. et al. A transmission-virulence evolutionary trade-off explains attenuation of HIV-1 in Uganda. eLife 5, e20492, doi:10.7554/eLife.20492 (2016).
Analyzing Chemotaxis to Understand How Cells Really Find Infections
Prof Rob Insall
It is now clear that immune cells like neutrophils and T cells locate the sites of infection using chemotaxis - migration steered by diffusing signals. However, simple chemotaxis is very inefficient, and immune cells need to reach infections quickly and efficiently. In this project we will test how neutrophils create their own chemoattractant gradients, and sharpen ones that already exist, using cell-surface proteases. We will test cells navigating through microfluidic mazes (see Tweedy et al., (2020) Science, 369(6507), eaay9792) in response to chemokines such as interleukin-8, and use CRISPR to find how different proteases control the accuracy of cell movements.
Predicting the molecular targets of ivermectin in human and veterinary helminths
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 . 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 . 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 .
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.
- 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.
Effect of improved water access on Schistosoma mansoni transmission
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.
Understanding host-parasite interactions using single cell transcriptomic analysis of trypanosome parasites and their mammalian host.
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.
Identifying the location of cytokine producing immune cells following influenza virus infection
Interferon-g is a key cytokine in the defence against respiratory virus infections, although a dysregulated inflammatory response can lead to immunopathology. Understanding which cells produce IFN-g following viral infection can aid in the identification of cellular targets to either enhance or reduce cytokine production.
Using a mouse model of influenza A virus (IAV) infection and transgenic mice that report IFN-g production, we have defined which immune cells produce IFN-g at different time points following infection. IFN-g is produced first by a range of innate cells including Natural Killer cells and Innate Lymphoid Cells while CD4 and CD8 T cells dominate the response at later timepoints.
This mini-project will advance our understanding in two ways. First, by using flow cytometry to define in more detail the identity and phenotype of IFN-g producing immune cells during IAV infection. Second, by defining the location of these IFN-g+ immune cells by immunofluorescence. This will address whether IFN-g producing immune cells are restricted to particular areas in the infected lung or can be found throughout the tissue. The student will be trained how to perform flow cytometry and fluorescent microscopy and how to analyse these data using Flowjo, ImageJ and Velocity software.
Helminth modulation of the intestinal epithelium in immunity and repair
The intestinal epithelium is not only an absorptive surface and barrier, but plays a critical role in sensing and repelling pathogens, and is an active participant in repairing damage following resolution of infection.
Some parasites, in particular helminth worms, exert immune suppressive effects resulting in, for example, an expansion of regulatory T cells in the tissues .
We have recently discovered that they also modulate the intestinal epithelium, altering the balance of specialised cell types that are required for parasite expulsion, and promoting proliferation (repair) of the epithelium at the expense of differentiation; this was also reported by an independent group .
These effects can be reproduced with soluble products released by the helminths, suggesting that epithelial homeostasis could be restored by blockade of these products, or additionally that some helminth molecules could be therapeutically useful in accelerating epithelial regeneration.
In this project, in vitro models of epithelial differentiation will be studied using organoid cultures, treated with helminth products; fractionation and mass spectrometry will be used to identify candidate modulators which will be expressed as recombinant proteins for validation and further study.
These proteins will be tested in in vivo models of intestinal pathology such as colitis, for their ability to promote repair and recovery, and also in a model of asthma to ascertain if effects are also apparent on airway epithelial cells.
Depending on the nature of the identified proteins, it may be possible to conduct mechanistic studies, identifying host receptors and signalling pathways, and to develop them as vaccine targets aiming to block the ability of parasites to manipulate epithelial cell fate.
 Johnston, C.J.C., Smyth, D.J., Kodali, R.B., White, M.P.J., Harcus, Y., Filbey, K.J., Hewitson, J.P., Hinck, C.S., Ivens, A., Kemter, A.M., et al. (2017). A structurally distinct TGF-β mimic from an intestinal helminth parasite potently induces regulatory T cells. . Nature Communications 8, 1741.
 Nusse, Y.M., Savage, A.K., Marangoni, P., Rosendahl-Huber, A.K.M., Landman, T.A., de Sauvage, F.J., Locksley, R.M., and Klein, O.D. (2018). Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche. Nature 559, 109-113.
Host-pathogen interactions during chronic Helicobacter infection
Helicobacter species establish chronic infection of the gastrointestinal tract of their mammalian host. Although they have been associated with deleterious inflammatory responses under some circumstances, in most cases chronic infection is not accompanied by any clinical disease. Recent work has also indicated that chronic Helicobacter infections can induce immune regulatory responses that prevent chronic inflammation, suggesting that a beneficial equilibrium is reached. However, the host and microbial factors that govern how such a set point is established are not well understood. This project will use experimental infection with intestinal Helicobacter species as a model to identify host-pathogen interactions that regulate protective and pathogenic immune responses in the gut. A better understanding of these pathways may offer new insights into preventing harmful immune pathologies in the intestine, such as inflammatory bowel disease.
Defining the genetic signature of malaria transmission
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.
1. Brancucci NMB, et al. (2017) Cell 171:1.
2. Rutledge GG, et al. (2017) Nature 542(7639):101.
Prising open mechanistic understanding of antigenic variation in Trypanosoma congolense
All pathogens must survive immunity to prosper within and spread between their hosts. Antigenic variation (AV), involving surface antigen switching to evade adaptive immunity, is a particularly widespread survival reaction, found in viral, bacterial and eukaryotic pathogens.
In T. brucei, AV of its variant surface glycoprotein (VSG) coat has evolved remarkable mechanistic complexity, relying on a repertoire of >2000 VSG genes and ‘pseudogenes’ . Only a single VSG is expressed at a time on any given cell, since the sole transcribed VSG must reside in a ‘VSG expression site (ES)’, which is a telomeric, multigene, RNA Polymerase I-transcribed genome feature. However, T. brucei contains not just one VSG ES, but many. As a result, T. brucei may execute a switch in VSG coat composition in two ways: by silencing the actively transcribed ES and activating transcription from one other ES, or by recombination of a silent VSG into the active ES. These reactions appear mechanistically distinct, but such a view must be tempered by a lack of understanding of how they are initiated and regulated.
The ATR kinase is a known component of the eukaryotic DNA damage repair machinery, acting to signal DNA lesions and recruit the appropriate repair machinery. Our recent work has shown that RNAi-mediated depletion of T. brucei ATR not only increases DNA recombination but also leads to impaired VSG transcriptional control, indicating ATR is a key part of the AV signalling machinery . Though T. congolense employs AV in the mammal virtually nothing is known about the underlying mechanisms . In this project we will exploit these recent findings on T. brucei ATR to begin to explore AV in T. congolense, asking if the reaction employs recombination or transcription-based VSG switching. To do so, we will build on recent developments in genetic modification of T. congolense and establish ATR-targeting RNAi in the animal parasite. We will then use a combination of RNA-seq, proteomics and ChIP-seq to ask if loss of ATR produces effects on VSG expression and switching due to altered recombination, transcription, or both.
- McCulloch R et al. Microbiol Spectr 2015, 3:MDNA3-0016-2014.
- Black, JA et al (2020). Cell Reports 30, 836-851 e5
- McCulloch, R et al. (2017) Emerging Topics in Life Sciences 1 (6) 585-592;
Resolving the mechanisms whereby chronic infection drives accelerated co-morbidities - project entailing model and human comparator studies
There is increasing evidence that the presence of chronic inflammatory disease in turn promotes acceleration of other systemic disorders including heart disease, stroke risk, diabetes and metabolic syndrome. As such, in high value economic countries, many immune-mediated disorders lead to early death due to these so-called co-morbidities. In parallel in Lower Middle Income Countries (LMIC), there is a rapid expansion of non-communicable diseases including CV disease, cancer and diabetes such that these disorders are now a leading cause of death even in areas of high infectious burden. Moreover, there is increasing recognition of immune-mediated diseases in LMIC for reasons that are unknown at present. This project will explore the hypothesis that the presence of chronic infectious burden in LMIC leads to a dysregulated immune response that can accelerate the subsequent development of co-morbid states by driving distant inflammation in blood vessels, the metabolic system (e.g. adipose tissue) and the brain (e.g. depression). Moreover, it will look at novel immune mediated mechanisms whereby underlying infectious burden in earlier life can predispose to, and mechanistically explain the subsequent development of immune mediate disorders themselves. The fellowship will entail the use of state-of-the-art epigenetic, molecular and cellular assays, appropriate development of in vivo models as required, and reference to, and use of samples from human cohorts of patients in LMIC, especially with a focus in Malawi or Tanzania. The project will appeal especially to investigators who seek a translational element to their studies that have strong applied clinical relevance for humans with chronic diseases, underpinned by a robust training in molecular and cellular biology.
Recent reviews from our group to provide background:
1: Ferguson LD, Siebert S, McInnes IB, Sattar N. Cardiometabolic comorbidities in RA and PsA: lessons learned and future directions. Nature Reviews Rheumatol. 2019 Aug;15(8):461-474.
2: Nerurkar L, Siebert S, McInnes IB, Cavanagh J. Rheumatoid arthritis and depression: an inflammatory perspective. Lancet Psychiatry. 2019 Feb;6(2):164-173.
How do intestinal parasites direct the immune response?
Prof Simon Milling
Helminth infections remain common in less-industrialised countries, where they significantly damage the health of millions of people. Conversely, their absence from industrialised countries has been associated with immune system dysregulation, leading to the ‘hygiene hypothesis’. Many socioeconomically important parasites undergo life cycle stages in the intestine, and elicit strong immune responses that attempt to control the infections. These responses are characterised primarily by Th2-type T cell responses, which produce the cytokines IL-4, IL-15 and IL-13. Beyond initiation of Th2-type T cell responses, some parasites have also been described to have immunomodulatory abilities that are not fully understood.
My lab has a long-standing interest in understanding how dendritic cells (DCs) contribute to controlling intestinal immune responses. Previous PhD students have developed tools for tracking the DCs that migrate from the intestine to the specific lymph nodes where they interact with naive T cells, and for purifying and for analysing the functions of these migratory DCs (See papers by Houston, Mayer, Andrusaite below). We are currently building on our success with bulk RNAseq and microarray analyses of migrating DCs, and have received funding to perform single cell sequencing analysis of intestinal migratory DCs. These DCs will be purified from animals after intestinal delivery of Schistosome Egg Antigen, or after infection with Heligmosomoides polygyrus, and the antigen-carrying DCs compared with DCs carrying antigen delivered in control of Th1/17-polarising conditions. This PhD will build from this work, testing hypotheses associated with understanding the signals delivered from the intestine to polarise immune responses in vivo.
The specific experiments will be designed in collaboration between the student, PI and postdoc, and will depend on the student’s specific interests.
Students undertaking this PhD or mini-project can expect to receive excellent training in understanding the immunology of the intestinal immune response at a cellular and molecular level. They will be provided opportunities to become expert in flow cytometric analysis and sorting, molecular biology (qPCR, ELISA), in analysing complex gene expression datasets (with bioinformatic support), and in working with animal models of infection.
The project will be based in the Sir Graeme Davies Building on the Gilmorehill Campus and will involve close interactions both with local groups working in mucosal Immunology and parasitology (Maizels., Maloy, and Perona-Wright), and with external collaborators, including Andrew MacDonald at the University of Manchester.
Houston SA, Cerovic V, Thomson C, Brewer J, Mowat AM, Milling S. The lymph nodes draining the small intestine and colon are anatomically separate and immunologically distinct. Mucosal Immunol. 2016 Mar;9(2):468-78. doi: 10.1038/mi.2015.77. Epub 2015 Sep 2. PMID: 26329428.
Mayer JU, Brown SL, MacDonald AS, Milling SW. Defined Intestinal Regions Are Drained by Specific Lymph Nodes That Mount Distinct Th1 and Th2 Responses Against Schistosoma mansoni Eggs. Front Immunol. 2020 Oct 23;11:592325. doi: 10.3389/fimmu.2020.592325. PMID: 33193437; PMCID: PMC7644866.
RNA viruses, molecular evolution & recombination
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.
New threats from old friends?
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
Dissecting the divergent and essential mitochondrial complex II of malaria and Toxoplasma,
Apicomplexan parasites, including Plasmodium and Toxoplasma, cause diseases of global importance such as malaria and toxoplasmosis, for which drugs are still needed. The apicomplexan mitochondrial electron transport chain (mETC) is important for parasite energy generation and is a proven target for antimalarial and anti-toxoplasmosis drugs (for example, Atovaquone). Recent discoveries suggest the apicomplexan mETC is highly divergent from their mammalian hosts (PMID: 31381948). For example, mETC complexes IV and V have many apicomplexan-specific components (PMID: 30204084; 30204085 30005062). One of the mETC complexes, complex II, also known as succinate dehydrogenase, is an important entry point of electrons into the mETC, as well as a TCA cycle enzyme.
Despite its importance for parasite energy metabolism complex II composition and function are poorly understood in apicomplexans. One barrier to study complex II has been the lack of tools to isolate the complex for proteomic 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: 31339607) and via the application of advanced proteomics methods (Maclean et al; unpublished. Draft available on request). We identified complex II 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 this complex works and potentially how inhibitors might interfere with its 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:
Wolbachia-mosquito-arbovirus interactions and dengue transmission blocking in Aedes aegypti
Wolbachia are intracellular, maternally inherited bacterial symbionts found in many insects.
The mosquito Aedes aegypti is naturally Wolbachia-free, but artificially introduced Wolbachia can completely block transmission of the viruses it transmits such as dengue and Zika.
Wolbachia also induce cytoplasmic incompatibility (CI), a sterility mechanism that allows it to rapidly invade insect populations.For these reasons Wolbachia are becoming important biocontrol of dengue.
A better understanding of the mechanistic basis of viral inhibition is needed in order to be able to deploy Wolbachia optimally for virus control.
Proteomic and functional assays comparing Wolbachia-carrying and Wolbachia-free Aedes aegypti cells have revealed that Wolbachia perturbs cholesterol and lipid transport in the cell, and this impacts viral replication.
The aims of the project will be to investigate how perturbations in lipid transport / homeostasis vary between Wolbachia strains and mosquito host species, and how their effects may differ between viruses.
We have already generated Aedes cell lines carrying a number of different Wolbachia strains, which reach different densities and show varying degrees of virus inhibition.
Various manipulations of regulatory genes will be carried out in these cell lines, the effects on lipids and Wolbachia density examined, and whether replication of dengue and Zika can be rescued.
Elevational gradients in infection and immunity in vampire bats
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.
Identification of new and emerging viral genomes in Uganda
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.
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.
Validating protein kinases as a target for next generation anti-malarial drugs
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).
Identification of the molecular target of a species-specific antibiotic active against drug-resistant Pseudomonas aeruginosa
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.
ABC transporters and commitment to sexual development in malaria parasites
Rotation programme abstract:
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.
Kent et al (2018) Nature Microbiology PMID 30177743
Lee, R.S. Waters, A.P.* & Brewer J.M.* (2018) Nature Communications. PMID 29703959.
Identifying the antiviral defences that constrain the emergence of coronaviruses in human populations
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.
TRIM69 Inhibits Vesicular Stomatitis Indiana Virus. Rihn SJ, Aziz MA, Stewart DG, Hughes J, Turnbull ML, Varela M, Sugrue E, Herd CS, Stanifer M, Sinkins SP, Palmarini M, Wilson SJ. J Virol. 2019 Sep 30;93(20). pii: e00951-19. doi: 10.1128/JVI.00951-19. Print 2019 Oct 15.
Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses. Shaw AE, Hughes J, Gu Q, Behdenna A, Singer JB, Dennis T, Orton RJ, Varela M, Gifford RJ, Wilson SJ, Palmarini M. PLoS Biol. 2017 Dec 18;15(12):e2004086. doi: 10.1371/journal.pbio.2004086. eCollection 2017 Dec.
Investigating the interactions of a novel tick-borne Banyangvirus with the mammalian innate immune system.
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.
- 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).
Drosophila as a model to study local and systemic impact of intestinal pathogenic infection
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
Iron and immunity in Toxoplasma gondii infection
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.
How malaria parasite gene expression adapts to mosquito host: in-depth analysis of Plasmodium berghei transcriptome during the initial stages of transmission
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.
High Dimensional 3D Imaging of Influenza Draining Lymph Nodes to Understand the role of Myeloid Organisation in Effective T-cell priming
Dr Ed Roberts
The lymph node is highly organised containing an array of immune cells coordinating to drive appropriate immune priming despite being distal to the site of infection. During Influenza A infection several DC subsets are thought to be involved in priming T-cells, however due to the complexity of manipulating DC subsets, the roles and importance of these subsets is unclear. Untangling the importance of different interactions in the lymph node will be important for us to rationally improve responses to vaccines and against tumours both of which are suboptimal currently. Thus, we aim to understand the initiation of effective T-cell immunity during infection and determine which adjuvants most closely recapitulate this. In the longer term we aim to apply this to not only improve vaccine adjuvant choice but to use those same approaches to improve sub-optimal tumour responses to increase the efficacy of immunotherapy.
In this project we will use our newly generated fluorescent influenza A virus to track viral derived antigens from the lung to the lymph node by flow cytometry. We will couple this with highly multiplexed 3D imaging of cleared lymph nodes to map antigen bearing DC subsets and their interactions with antigen specific T-cells through an infection time course. Using spatial analytic techniques we will seek to identify microenvironmental niches characteristic of anti-influenza immune responses and compare these to what is obtained with different vaccination approaches. Longer term this approach will be applied to understand how signals from different types of infection direct lymph node organisation to drive appropriate immune responses.
High-dimensional investigation of the blood-brain barrier in cerebral malaria.
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.