Nematode surface properties and genetics, drug sensitivity and bacterial infection

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

The external surface of an animal is a major determinant of how it interacts with its surroundings. Maintaining surface integrity is essential for normal development and survival, so the surface layers provide essential protection, but they also constitute a vulnerable area for attack by pathogens. We are examining how bacteria infect the outside of an animal, and we use bacterial infections to determine how the outermost layers of this animal develop and maintain a permeability barrier. We are also studying what these outer layers are made of, and how the animal responds and defends itself against bacterial infection.
We make use of a very simple animal called C. elegans, a tiny nematode worm which normally lives in the soil. This worm has provided a phenomenally useful laboratory system for studying many different biological and medical problems, and consequently well over 500 research groups around the world now use it as an experimental system. Some of its experimental advantages are that it is very cheap and easy to grow, with a 3 day generation time allowing for rapid genetic experiments, and it is wholly transparent, so that development and disease can be looked at directly in the living animal. Latterly, it has been increasingly used for studying processes of bacterial and fungal disease.
We have discovered several natural bacterial strains which either damage or efficiently kill C. elegans worms, by infecting the outside of the animal. Using these bacteria, we can select for genetically altered worms that are resistant to infection, and have found dozens of genes that are involved in the first (infection) or subsequent steps in disease. Many of these genes turn out to be needed to make the impermeable surface coat of the worm, and they provide us with a route to establish what constitutes this outer layer, and how the pathogenic bacteria can attach to it. Other genes are involved in later disease stages, and provide information on how the presence of pathogenic bacteria is detected, and what kind of antibacterial defenses are used. Part of the worm's defense response involves a striking enlargement of certain cells within the animal. Studying the molecular mechanism of how these cells swell up provides basic insight into how cells can change their shape and size during normal development.
There are additional practical reasons for looking at permeability and infection in this animal. One reason is that the worm provides an easy way of screening for new antibiotics and drugs. Because the animals are so small and so easily handled, it is possible to test hundreds or thousands of candidate drugs for effects, much more cheaply, speedily and ethically than by using mice, for example. Novel antibiotics and anti-ageing drugs, and compounds that would ameliorate Alzheimer or Parkinson disease, are being sought by this means. However, many candidate drugs that would work on humans are likely to be ineffective in C. elegans, because they fail to permeate into the animal. This problem may be overcome by using some of the mutant worms we work with, because their surfaces are leaky and consequently they are much more sensitive to drug treatments. We intend to develop a range of drug-sensitive worm strains that will greatly increase the scope of these drug screens.
A second reason is that many nematode species of nematode are important plant and animal parasites, of great economic importance in agriculture and forestry, and some are significant human pathogens, especially in the developing world - at least a billion people suffer from nematode infections. Examining the vulnerabilities of C. elegans, a conveniently harmless and non-parasitic nematode, to nematode-specific pathogens, may offer new routes to biological or chemical control of nematodes. There is a severe lack of good anti-nematode drugs, both in medicine and in agriculture.

Technical Summary

The external surface of an animal provides a protective barrier, allowing normal development, physiology and interactions with the environment, and also constitutes a major area for attack by pathogens. This proposal explores nematode surface coat development, biochemistry and interaction with pathogens, using the nematode C. elegans. Much of the proposal stems from discovery of two virulent and complementary bacterial pathogens for C. elegans called Verde1 and Verde2. These provide selective tools for exploring the genetics of surface coat and bacterial interaction.
We propose investigations in six areas:
1. Surface mutant genetics, carrying out selections and RNAi screens for genes affecting bacterial infection and surface permeability. Suppressor screens for modifiers of existing mutants will be performed, to reveal gene interaction networks.
2. C. elegans drug sensitivity. Many of the surface mutants are increased in drug sensitivity, as a result of increased permeability and drug uptake. We will investigate drug permeation, aiming to improve C. elegans-based drug screens.
3. Nematode surface biochemistry. We will analyse the carbohydrate and protein composition of surface coat, use tagged transgenes and mass-spectrometry.
4. Pathogen genetics. We will determine genome sequences for Verde1 and Verde2, and investigate their virulence and adhesion properties.
5. Cellular swelling and developmental plasticity. Infection by Verde2, as with a previously studied pathogen Microbacterium nematophilum, induces a protective cellular swelling reaction in rectal epithelial cells. We will investigate the cell biology, mechanism and function of this morphological change.
6. Analysis of pathogen detection and innate immune changes. Both Verde1 and Verde2 induce specific defense responses in infected worms. We will investigate the signal transduction pathways involved, and the nature of possible bacterial triggers for these immune reactions

Planned Impact

The proposed research has several possible practical applications in terms of improving human health and welfare.

1. The exploration of drug uptake in C. elegans, and the development of new drug-hypersensitive strains, has consequences for high-throughput screens for pharmacology and antibiotic development. Many chemicals do not permeate significantly into wildtype C. elegans, but they are likely to permeate much more effectively into hypersensitive strains. C. elegans is nevertheless being used to screen for novel antibiotics active against bacteria or fungi, and for drugs active against human neurodegenerative conditions, either by using existing mutants or by 'humanizing' worms with the appropriate human gene constructs (eg beta amyloid). C. elegans is also a premier model for studying ageing, and we have shown that rapamycin is able to prolong the lifespan of our hypersensitive mutant worms although rapamycin fails to work on wildtype worms. Creation of a range of different strains with varied and defined drug uptake properties will substantially enhance the usefulness of C. elegans as a system for drug development in diverse areas. Examining how to further increase permeation, for example by detergent treatment, may provide ways of increasing efficacy of nematicides. We will pursue development of optimal nematode hosts and protocols for drug screening, in collaboration with David Sattelle (University of Manchester).

2. Better understanding of surface coat composition, formation and turnover in C. elegans, as a paradigm nematode, has implications for interactions between human parasitic nematodes and the immune system. Many such parasites are able to avoid or reduce immune clearance, partly because of their specialized surface coats, but the coat is a poorly understood layer in any nematode.

3. Second, investigation of the nematode killing or inhibiting properties of the Leucobacter strains Verde1 and Verde2 has potential for the control of nematode parasites in general. As parasites of agriculturally important plants and animals, nematodes inflict huge economic burdens on the world; as parasites of humans, nematode pathogens are responsible for disease in billions of people. Better understanding of nematode-specific susceptibilities and new ways of killing nematodes may allow development of new nematicides, which are in very short supply. The Leucobacter strains may also allow development of biological control agents, especially if it proves possible to expand their host-range. We will collaborate with Keith Davies (BBSRC Rothamsted) in testing action on plant parasitic nematodes.

4. New targets for nematicide development: Some of the proteins we are studying, such as the predicted glycosyltransferase BUS-8 and the galactofuranose GLF-1, are effectively essential for the survival of C. elegans (and presumably other nematodes as well) but appear largely specific to the nematode phylum, so they present attractive targets for the development of drugs selectively acting on nematode parasites.
We anticipate that our screens and selections will identify further nematode-specific targets.

5. Better understanding of the ways in which bacteria adhere to target surfaces is fundamental to the understanding of many different kinds of bacterial infection, and also to the formation of bacterial biofilms. Most infections begin with the adherence of a pathogen to a biological surface, but the chemistry of the initial adherence is often mysterious. Moreover, adherence and accumulation of bacteria to a surface may lead to the formation of a biofilm, which can have disease consequences. Biofilms are also economically important in many contexts, such as marine fouling. Our proposed research should allow genetic dissection and better understanding of adhesion simultaneously on the bacterial side and on the host side.

Publications

10 25 50

 
Description Faucett Catalyst Grant
Amount $15,000 (USD)
Organisation Genetics Society of America 
Sector Private
Country Unknown
Start 09/2013 
End 09/2015
 
Description MRC Programme Grant
Amount £1,866,841 (GBP)
Funding ID MR/J001309/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2012 
End 03/2017
 
Title Drug-sensitive nematode strains 
Description We have developed a series of mutant strains of the nematode C. elegans with greatly increased sensitivity to many different drugs, arising from altered cuticle permeability. 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact General improvement in the ability to use C. elegans for drug screening and pharmacological manipulation. 
 
Title Nematode bacterial pathogens 
Description Strains of novel bacterial pathogens of nematodes 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact None as yet 
 
Title Nematode mutant strains 
Description Characterized mutant strains of the nematode C. elegans 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Analysis of C. elegans responses to Yersinia pestis, causative agent of plague 
 
Description Investigation of nematode Patched-Related gene functions in infection and development 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution We have shown that our wildtype transgene constructs rescue the developmental lethality of a ptr-15 deletion generated by the collaborator, but that intestine-specific expression of this gene (using her constructs, injected by us) fails to correct the immunodeficiency exhibited by a viable missense ptr-15/bus-13 mutant.
Collaborator Contribution CRISPR knockouts, tissue-specific transgene constructs, genetically suppressed lines, intellectual input.
Impact Poster presentation at international meeting.
Start Year 2013
 
Description Nematode glycobiology 
Organisation Food and Drug Administration (FDA)
Department Centre for Biologics Evaluation and Research
Country United States 
Sector Public 
PI Contribution Provision of mutant and transgenic nematode lines, phenotypic investigation by microscopy and cell biology
Collaborator Contribution Mass-spec analysis of nematode glycans
Impact PMID: 20385555
Start Year 2008
 
Description Yersinia biofilm formation 
Organisation University of California, San Francisco
Department Department of Cell and Tissue Biology
Country United States 
Sector Academic/University 
PI Contribution Molecular cloning, expression analysis and phenotypic characterization of genes affecting nematode surface properties
Collaborator Contribution Testing of pathogenicity and biofilm formation by Yersinia bacteria
Impact PMID: 20980242
 
Description biopterin cofactor synthesis 
Organisation University of San Diego
Department Department of Biology
Country United States 
Sector Academic/University 
PI Contribution Generation of biosynthetic mutants, analysis of phenotypes and genetic interactions
Collaborator Contribution Enzyme and catecholamine assays, behavioural analysis.
Impact None
Start Year 2012
 
Description cholinergic neurogenetics 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution Genetic data, mutant strains
Collaborator Contribution Molecular and physiological data
Impact PMID: 16604067
 
Description ether lipids and alkylglycerol monoxygenase 
Organisation Medical University of Innsbruck
Department Division of Biological Chemistry
Country Austria 
Sector Academic/University 
PI Contribution Isolation and analysis of mutants, analysis of phenotypes, generation of transgenic lines, provision of material for enzyme assays.
Collaborator Contribution Enzyme assays, heterologous expression of nematode genes in mammalian cells
Impact None
Start Year 2012
 
Description Art and Science in Oxford 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I organized a public meeting in Jan 2006, on Art and Science in Oxford, in relation to the Salt Bridges Arts Programme for the New Biochemistry building. This included description of my research.

Increased awareness of biochemical and medical research, and its relationship to the arts. Increased funding for art/science projects.
Year(s) Of Engagement Activity 2006
 
Description School Visit (Wycombe Abbey) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Schools
Results and Impact 40 pupils attended, with subsequent questions and discussions.

Substantial interest and enthusiasm was expressed.
Year(s) Of Engagement Activity 2013