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 School of Biodiversity, One Health and Veterinary Medicine, and with the MRC-University of Glasgow Centre for Virus Research.
Discovery of antimicrobial drug targets using metabolomics
The requirement for new antimicrobial agents is of increasing urgency. Screening for new compounds that kill microbes is bringing new chemicals forward. However, beyond simply killing the microbial organisms it is also necessary to understand how compounds selected in this manner exert their activity. Working with the parasitic protozoan Leishmania, which afflicts millions of people in the tropics and sub-tropics, we have identified numerous compounds that are toxic to the parasite. In this rotation project, novel agents that have been shown to kill leishmania parasite will be applied to parasites and changes to cellular metabolism that result from this exposure will be ascertained using untargeted metabolomics technology. If compounds target specific enzymes, these will be identified based on the changes to metabolism associated with exposure to the drugs. Targets will then be verified using genetic modification of parasites with CRISPR-cas9 technology to confirm that putative targets are essential and that their over-expression can diminish sensitivity to the drugs.
Understanding the genomic context of noncoding RNA in the schistosome genome
Schistosomiasis is a major neglected tropical disease caused by blood flukes of the genus Schistosoma. Fundamental research into schistosomiasis is needed to inform the search for new approaches to disease intervention. Schistosoma mansoni is one of the clinically most important species, and the most widely studied. A high-quality genome reference has provided the foundation for large-scale studies of schistosome biology. Other schistosome species are less well studied, but they differ in their life cycles, within-host tropisms (and subsequent pathologies), prevalence and geospatial distributions. Reference genomes for 19 species from the Schistosomatidae family have now been sequenced.
Preliminary comparisons have revealed a striking degree of conservation at the level of gene repertoires, suggesting that much of the phenotypic differences between these species is driven by differences in expression of highly similar gene repertoires. Regulatory non-coding RNAs (ncRNAs) play an important role in regulating gene expression in many organisms, but their contribution to schistosome diversity has not been explored. In this project you will align the genome sequences of diverse schistosome species to define evolutionary conserved regions. Using existing data and structural approaches to predicting potential ncRNAs in these genomes you will explore the relationship between the evolution of ncRNAs and their expression in existing single-cell RNAseq data to develop a first understanding of their role in the diversity of schistosomes.
(Co-supervised by James Cotton)
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
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.
REFERENCES
- 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
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.
Microbiome, metabolome, epigenome: How do microbiota regulate host ageing?
Globally, human health is compromised by our ageing populations, which erodes population health through a multitude of age-related diseases. Healthy ageing is modulated strongly by gut microbiota, but we do not know how or why. However the interactions between host and microbe are conserved throughout animals, including humans, which means that we can use simple animal models to understand fundamental microbial mechanisms that underpin human health. In this project you will use a simple but powerful system to investigate how bacterial genetics influence host epigenetics, and what those epigenetic changes mean for host lifespan and metabolism.
You will use such a powerful but simple model - fruitflies - to dissect the molecular basis of the microbial influence on host health. This system offers total control of all aspects of the microbiota, diet, and host molecular function, and in that context we can identify bacterial genes that influence host function and health. Your rotation will likely include investigation of histone protein modifications via protein biochemistry, bioimaging, and gene expression assays. A full multidisciplinary PhD project would additionally entail studies of age-related health, genetics (in both bacteria and flies), metabolism & nutrition, bacterial genetics, and 'omics; with the long-term goal of identifying fundamental processes that occur in all animal hosts.
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 African malaria vectors
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.
References
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 humans and significant production losses in economically crucial livestock species (e.g. cattle, sheep and goats) globally. There is evidence that C. burnetii genotypes vary in their pathogenicity and epidemiology, but robust data on data the distribution and clinical and production significance of these genotypes in different global settings is lacking.
- burnetii is endemic in Tanzania, where C. burnetii infection, also known as Q fever, causes substantial burdens of human illness. Serological and molecular testing reveals that C. burnetii exposure is common in livestock and bacterial carriage and shedding was detected goats, cattle and rodents providing evidence of the complex multi-host epidemiology of this important pathogen in Tanzania.
- Generation of diagnostic test data on bacterial shedding to improve understanding of transmission routes and processes within and between animal populations (including environmental persistence)
In the UK context, recent legislative changes requiring national reporting of C. burnetii infections in livestock combined with evidence of widespread infection in dairy cattle across the UK has identified crucial gaps in our current understanding of the epidemiology and impacts of C. burnetii on animal health and productivity.
Despite growing recognition of the widespread distribution of C. burnetii in ruminant populations globally and the zoonotic impacts of Q fever in many global contexts, considerable gaps remain in our understanding of the relationships between diagnostic test outcome data and epidemiological parameters, the influence of variation in genotypes on epidemiology and pathogenicity of Q fever. This lack of knowledge impedes current efforts to evaluate candidate surveillance and control measures that are applicable and appropriate for these different epidemiological contexts.
This project focuses on the generation and analysis of molecular detection and genotypic data to advance our understanding the epidemiology of C. burnetii in Tanzania and the UK. The project will build on access to previously collected samples from humans, livestock and wildlife populations from Tanzania as well as prospective sampling in UK livestock populations.
Specific objectives may vary depending on the candidates’ preferences, with scope for project development in multiple directions. Potential areas of focus include:
- Characterisation of genetic variation in burnetii - detected from multiple hosts sampled in both the UK and Tanzania
- Quantification of the livestock production and human health impacts of Q fever and to inform evaluation of context appropriate disease control interventions
Training opportunities will span diagnostic technologies and epidemiological analyses with opportunities to work within an interdisciplinary team on this One Health challenge. Work will include colleagues at the School of Biodiversity One Health and Veterinary Medicine.
References:
Pisharody et al. (2021) Incidence Estimates of Acute Q Fever and Spotted Fever Group Rickettsioses, Kilimanjaro, Tanzania, from 2007 to 2008 and from 2012 to 2014. Am J Trop Med Hyg doi: 10.4269/ajtmh.20-1036.
Salifu, S. P., Bukari, A. A., Frangoulidis, D. & Wheelhouse, N. Current perspectives on the transmission of Q fever: Highlighting the need for a systematic molecular approach for a neglected disease in Africa. Acta Trop 193, 99-105 (2019).
Vanderburg S., Rubach, M.P., Halliday, J.E.B., Cleaveland, S., Reddy, E.A. & Crump, J.A. (2014) Epidemiology of Coxiella burnetii infection in Africa: a OneHealth Systematic Review. PLoS Negl Trop Dis. 8 (4): e2787.
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).
Temporal regulation of influenza virus infectivity’
Like many medically important viruses, influenza viruses are transmitted through virions that can vary markedly in their form and composition. We have previously shown that the proteins incorporated into influenza virions can be determined by the host species, and we now have data showing that the virions shed from a single infected cell change as the infection progresses. Infected cells shed influenza virions for hours, and virions shed at later timepoints incorporate exponentially increasing amounts of a viral immunosuppressive protein. Preliminary data suggest that this makes ‘late’ virions more able to overcome the defences of new host cells, which could help to overcome the effects of paracrine immune signalling. In this rotation project, you will use virology, cell biology and flow cytometry methods to test whether influenza is able to use its flexible architecture to increase in infectivity as an infection progresses.
Analyzing Chemotaxis to Understand How Cells Really Find Infections
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 [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.
Effect of improved water access on Schistosoma mansoni transmission
Dr Poppy Lamberton and Dr Stephanie Connelly
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 [1].
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 [2].
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.
[1] 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.
[2] 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.
References:
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
Understanding how DNA replication programming drives adaptive genome variation in African trypanosomes and Leishmania
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.
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?
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.
References:
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.
Understanding interactions among respiratory viruses
The human respiratory tract hosts a community of viruses that cause outbreaks, epidemics, and pandemics. Respiratory viruses have been traditionally studied in isolation and we know very little about the biological and epidemiological consequences of their interactions. However, we have shown that the interplay among respiratory viruses has an impact in their epidemiology (Nickbakhsh et al., 2019). The aims of this project are i) to identify previously unknown interactions among respiratory viruses; ii) to characterise the nature and mechanisms by which viruses interact; and iii) to integrate the impact of virus-virus interactions from the cellular to the population level. We will perform experimental infections of air-liquid interface cellular cultures representing the human respiratory tract and characterise the infection and co-infection phenotype of key respiratory viruses as we have previously shown (Dee et al., 2021). To study virus interactions at the cellular level we will combine classical virology, transcriptomics and image analyses, whereas to study changes in virus structure we will use cryo-electron microscopy (Haney et al., 2021). To estimate the population impact of the observed virus-virus interactions we will perform mathematical modelling (Dee et al., 2021; Nickbakhsh et al., 2019). This programme will provide fundamental new information on the complex biology of multi-pathogen systems and inform the design of control interventions. It will also change future research approaches to study viral infections.
References
Dee, K., Goldfarb, D. M., Haney, J., Amat, J. A. R., Herder, V., Stewart, M., . . . Murcia, P. R. (2021). Human rhinovirus infection blocks SARS-CoV-2 replication within the respiratory epithelium: implications for COVID-19 epidemiology. J Infect Dis. doi:10.1093/infdis/jiab147
Haney, J., Vijayakrishnan, S., Streetley, J., Dee, K., Goldfarb, D., Clarke, M., . . . Murcia, P. R. (2021). Generation of chimeric respiratory viruses with altered tropism by coinfection of influenza A virus and respiratory syncytial virus. bioRxiv.
Nickbakhsh, S., Mair, C., Matthews, L., Reeve, R., Johnson, P. C. D., Thorburn, F., . . . Murcia, P. R. (2019). Virus-virus interactions impact the population dynamics of influenza and the common cold. Proc Natl Acad Sci U S A. doi:10.1073/pnas.1911083116
Describing genomic fragment of a novel malaria genome
My lab is interested in the evolution and host adaptation of the malaria parasite. We sequenced several genomes of primate malaria. In one of the projects, we found multiple infections with a little described malaria parasite.
We found traces of DNA and assembled a few short contigs from this plasmodium genome found in Gorilla. It seems to be similar to human malaria. Unfortunately, little is known about this species, apart from some polymorphisms in the mitochondria.
In this in silico project, the student will analyse DNA traces and build phylogenetic trees to place it within the plasmodium phylogeny. Next, the aim is to scan other datasets to establish if more events can be found.
The aim is to describe this new species as good as possible.
It is of advantage to have knowledge in Linux or phylogeny.
Hitting the sweet spot: metabolic regulation of the immune response to infection
Every immune response has a sweet spot, at which the immune response is as effective as possible while causing minimal collateral damage. Understanding the regulatory mechanisms that set this balance is an important step in designing therapeutic strategies that artificially enhance or suppress immune responses. Our team focuses on the regulation of immunity to infection. We are particularly interested in the factors that regulate the balance between effective immunity and immunopathology, including the ways that (1) tissue location, (2) site-specific fuel sources and (3) cell-cell signalling networks can influence the metabolism, activation and function of immune cells at their point of contact with incoming pathogens.
The research questions involved in a PhD project will be designed with the interested PhD candidate, to include the student’s specific interests. The questions will include a focus on immunometabolism and tissue-based immunity, in vivo, in the response to infection. We use models of viral, bacterial and parasitic infections (influenza, Salmonella, and helminth parasites), often comparing Th1, Th2 and Th17 responses in single and dual infections.
Our lab is based in the Sir Graeme Davies Building on the Gilmorehill Campus, and we work happily and closely with several other in vivo immunology groups, including those of Simon Milling, Kevin Maloy, Ed Roberts, Rick Maizels and Donal Wall. Our emphasis is on in vivo models of infection, and our staff and students become highly skilled in flow cytometry, cytokine analyses, fluorescent imaging, transcriptomics, metabolomics and gene manipulation.
Genetic control of human malaria parasite growth rates
The aim of this project is to identify the genes in the human malaria parasite Plasmodium falciparum that determine differential rates of intraerythrocytic growth.
There are over 220 million cases of malaria caused by P. falciparum per year, and around 400,000 people die of severe forms of the disease. Identifying and targeting better health care to those individuals most at risk of severe disease and death is a priority to reduce mortality rates, but at present there is no clear marker, either in the host or parasite, associated with increased risk of mortality. Higher parasite growth rates in the blood, leading to increased parasite burdens, are associated with higher morbidity and mortality [1,2]. Previous work in my lab using a quantitative trait locus analysis [3] in a genetic cross between two P. falciparum clones identified genomic regions (qtl) contributing to asexual growth rate differences that differ in their invasion efficiency.
We have very recently culture-adapted a set of new parasites from people living in Mali. Some children presented with a form of severe malaria known as hyperparasitaemia, where there are very high numbers of parasites in the blood, possibly as a result of fast growth- but this is not understood. During the rotation project, you will characterise the growth rates of these parasites and analyse their variation in the genes within the qtl regions, and see if variation in these genes correlates with high growth rates. Is there good evidence to link growth rate with the occurrence of hyperparasitaemia?
The work would involve culturing the new malaria parasites, and measuring their growth rates (length of intra-erythrocytic cycle, number offspring per cycle, invasion efficacy). This would be coupled with some bioinformatics (sequencing). The project has plenty of scope to develop into different areas to explore these novel isolates,
References
- Chotivanich K, Udomsangpetch R, Simpson J, Newton P, Pukrittayakamee S, Looareesuwan S, White N (2000) Parasite Multiplication Potential and the Severity of Falciparum Malaria. J Inf Dis 181: 1206-1209.
- Tripathy R, Parida S, Das L, Mishra DP, Tripathy D, Das MC, Chen H, Maguire JH, Panigrahi P (2007) Clinical manifestations and predictors of severe malaria in Indian children. Pediatrics 120: e454-e460.
- Ranford-Cartwright LC, Mwangi JM (2012) Analysis of malaria parasite phenotypes using experimental genetic crosses of Plasmodium falciparum. Int J Parasitol 42: 529-534.
Characterising molecular and evolutionary properties of the virus-host interactome
MRC-University of Glasgow Centre for Virus Research (CVR)
E-mail: david.l.robertson@glasgow.ac.uk, @robertson_lab
Viruses are dependent on a host cell to replicate and do this by exploiting the molecular systems of that cell, using so-called host-dependency factors. In turn infection triggers an anti-viral response that’s evolved mechanisms to limit and learn how to counteract infection. These virus-host interactions form an intricate set of molecular – mostly protein-protein – interactions that can be represented and modelled using network graphs (Oyeyemi et al. 2015, PMID: 25431332; Ravindran et al., 2019, PMID: 30765882]). Such tools can help us represent and study a virus species’ life cycle and identify putative drug targets – either virus or host molecules – to interfere with infection as demonstrated recently for SARS-CoV-2 (Gordon et al., 2020, PMID: 32353859). Combining protein-protein interaction data with existing knowledge of host cell organisation, in particular molecular pathways (representation of information flow through directed molecular interactions linked to intra-cellular functions) with gene expression data from infected cells yields insights into the broader perturbation of infected host cells (MacPherson et al. 2010; PMID: 20686668). This project will involve using publicly available virus-host molecular interaction data (for example, available from VirHostNet, Viruses.STRING, IntAct etc.) and combine this with datasets available at the CVR, for example from bulk-RNASeq and single-cell transcriptome studies. Key aims will be to study 1/ the properties of host molecules, their immediate molecular partners and broader functional context and 2/ link these properties to the gene/protein content of evolutionary related host species used by the same virus species. A central hypothesis to test will be the extent to which the use of divergent evolutionary hosts by a virus species is mediated by the same molecular sub-systems. For example, arthropod-borne viruses (arboviruses) that successfully infect both insects and mammals could be used a model system to test the limits of host-dependency factors, for example linked to dengue virus infection. This will involve constructing bipartite networks and studying the conservation of pathway components/orthology between insects and mammals using public and CVR datasets. We hypothesise the requirement to use evolutionary divergent host systems places constraints on the virus that can be exploited for the design of anti-viral measures. The project can be tailored to candidates with a background in virology, biology, bioinformatics/computational biology, computer science, machine learning or mathematics.
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 COMPLEXS 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 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/
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.
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.
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).
How do interventions impact malaria vector communities?
Vector control interventions have been incredibly successful at reducing many mosquito populations. However, as these populations decline, ecological succession - the process of change in the species composition of a community over time - is likely to occur. For example, as one species nears elimination, it might leave (partially) empty niches, where other species could expand to. This is a big problem for malaria control because disease transmission is often maintained by a suite of mosquito species that may differ both in their efficiency at transmitting disease (either the same or a different disease) and response to interventions. Suppression or removal of one species could release others from competition thus triggering substantial changes in species abundance which could have positive or negative effects on the overall ability of the mosquito community to transmit malaria. In this project, you would develop models to quantify long-term changes in vector community dynamics driven by usage of insecticidal treated bednets in Africa using data collected during vector surveillance programs in Africa.
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.
Bacteria of the human gut microbiota as drivers of mammalian disease
Bacteria of the human gut microbiota as drivers of mammalian disease.
As chronic medical conditions such as type 2 diabetes and obesity increase in prevalence across the world, it is increasingly recognized that the gut microbiome may exacerbate such conditions through direct interference in host metabolism. Recent advances in imaging and small molecule identification have allowed us to understand the mechanisms behind such microbial influence, helping us to understand how microbes in the gut can undermine human health. Our own work has identified small metabolites produced by specific gut microbes that spread systemically in mice and directly interfere with lipid metabolism in multiple organs. The goal of this project is to understand the often poorly characterized microbes mediating these effects by focusing on; their capability to produce small metabolites, the function of these molecules in the host, and their ability to share or acquire the relevant genes through mobile genetic elements in the gut.
Relevant reading for the project includes;
Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication. Hulme et al. Science Advances 2020 doi: 10.1126/sciadv.aax6328
Microbiome-derived metabolites reproduce the mitochondrial dysfunction and decreased insulin sensitivity observed in type 2 diabetes. Ormsby et al. 2020 doi: 10.1101/2020.08.02.232447
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.
References:
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.
References
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.
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).
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
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.
Population structure during respiratory viral infection.
Evidence from a variety of sources supports the idea that acute respiratory viral infections form structured (that is, non-well-mixed) populations within a host. A study of influenza evolution in a human host showed evidence for a lack of reassortment, indicative of genetically distinct viruses occupying different niches within the host1. Evidence for diverging populations during within-host infection has been observed in long-term infections in SARS-Co-V-2 and influenza B2,3. Animal models have been used to identify multiple within-host bottlenecks arising during influenza infection4. The observation of within-host population structure is important as many mathematical approaches to understanding viral transmission and evolution5,6 work on the assumption that genome sequencing provides an unbiased, if noisy, description of a consistent underlying population. Recent experimental evidence suggests that population structure can be found at the smallest cellular scales7. We will employ a variety of tools to build an improved understanding of population structure during respiratory viral infections.
a) Use mathematical modelling to understand the cellular factors preventing mixed infections
Work from the Hutchinson group at the CVR has used in vitro models to identify patterns of superinfection exclusion during influenza infection: viral plaques grown in close proximity in cell culture do not co-infect cells to produce recombinant virus. We will combine mathematical models with experimental data to i) characterise the basic growth of viruses within plaques, building an understanding of why plaques grow and then cease to grow during experiments, and ii) extend this model to identify likely mechanisms behind superinfection exclusion.
b) Use evolutionary models to better understand within-host diversity in clinical cases of infection
We will use genome sequence data describing clinical cases SARS-CoV-2 and influenza infection to study within-host diversity. According to a model of mutation and genetic drift genetic diversity should accumulate over time, with variation being repeatedly observed through sequencing. We will use sequence data collected from multiple points in time to i) evaluate the extent to which observations are consistent with well-mixed populations, ii) infer, where possible, the underlying structure of viral populations, iii) build better models of how within-host diversity can be used to identify cases of transmission among data collected from clinical cases.
- Leonard, A. S. et al. The effective rate of influenza reassortment is limited during human infection. PLOS Pathogens vol. 13 e1006203 (2017).
- Kemp, S. A. et al. SARS-CoV-2 evolution during treatment of chronic infection. Nature 592, 277–282 (2021).
- Lumby, C. K., Zhao, L., Breuer, J. & Illingworth, C. J. R. A large effective population size for established within-host influenza virus infection. eLife vol. 9 (2020).
- Amato, K. A. et al. Influenza A virus undergoes compartmentalized replication in vivo dominated by stochastic bottlenecks. Nat. Commun. 13, 3416 (2022).
- Ghafari, M., Lumby, C. K., Weissman, D. B. & Illingworth, C. J. R. Inferring transmission bottleneck size from viral sequence data using a novel haplotype reconstruction method. doi:10.1101/2020.01.03.891242.
- Illingworth, C., Jr et al. Superspreaders drive the largest outbreaks of hospital onset COVID-19 infections. Elife 10, (2021).
- Sims, A. et al. Superinfection exclusion creates spatially distinct influenza virus populations. doi:10.1101/2022.06.06.494939.