Genetic, structural and functional analyses of flagellar glycosylation in epidemic Clostridium difficile strains

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Clostridium difficile is a bacterium that causes chronic diarrhoea and sometimes life-threatening disease (generally referred to as C. difficile infection or CDI) mainly in elderly and hospitalized patients. C. difficile is now the most prevalent and significant hospital acquired infection. Given the ability of C. difficile to evolve to be highly virulent coupled with the ageing population and increasing rates of hospitalization, the problem of CDI is likely to worsen. The reported incidence of CDI has risen dramatically over the last decade and is likely to be related to the emergence of aggressive sets of clonal strains such as the so-called PCR-ribotype 027 and 017 strains.

Disease causing bacteria often have flagella. This structure is important for motility and facilitates swimming towards nutrients and away from noxious agents, but also for intestinal pathogens such as C. difficile, flagella are used to penetrate the mucus lining of intestinal cells. A feature of several disease causing bacteria is that their flagella are decorated with sugars (glycans), which confer multiple properties such as improved ability of the bacterium to colonise and also to avoid host immune attack. Scrutiny of the genetic information of the aggressive 027 and 017 strains suggests that they have evolved distinct flagellar glycans that may partly explain why they are so problematic.

To help find ways to understand and prevent this disease, we will investigate how and why the 027 and 017 strains have specific flagellar glycans and the potential role of flagellar glycans in general in the survival and disease capability of C. difficile. A comprehensive understanding of flagellar glycosylation in C. difficile will strengthen our understanding of the microorganism and the disease as well as promoting the development of new treatments to prevent CDI.

Technical Summary

The current epidemic of C. difficile infection has been accompanied predominantly by the emergence of two clonal groups of PCR-ribotype 027 and 017 (A-B+) strains that are associated with increased transmissibility and disease severity. Recently, in collaboration with the WTSI, we sequenced the genomes of several 027 and 017 strains and identified a number of genes involved in flagellin glycosylation that are distinct from other C. difficile strains all of which appear to be glycosylated. Further studies have confirmed that flagellin from representative 027 and 017 strains are glycosylated and are structurally different to other C. difficile strains. The modification of flagella by glycosylation has been shown to be increasingly important in the virulence and host adaption of several bacterial pathogens. We hypothesise that flagellar glycosylation is important in C. difficile and that this may be especially relevant to the epidemic 027 and 017 strains that maintain distinct glycoforms. To determine the role and importance of flagella and flagellar glycosylation in C. difficile we will construct several 027 and 017 mutants to produce aflagellate mutants, glycosylation deficient mutants and glycosylation modified mutants. These distinct mutants will be rigorously characterised in several physical, in vitro and in vivo assays relevant to the life cycle of the organism. These studies will be augmented by structural analyses of the glycan component of the wild type 027 and 017 strains and the respective mutants. In this way we will dissect the process of flagellar glycosylation by a combination of genetic, bioinformatic and structural analyses enabling us to build up a comprehensive picture of the core and accessory genes involved in flagellar glycan biosynthesis. We will also determine if the same glycosylation pathway also modifies other surface structures. The characterisation of glycosylated surface exposed structures such as flagella should improve our knowledge of how C. difficile causes disease and could also prove useful in the development of diagnostic markers and the identification of therapeutic and vaccine targets.

Publications

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Cairns MD (2012) The continually evolving Clostridium difficile species. in Future microbiology

 
Description LY256: A novel and potent antibiotic for treating Clostridium difficile infection
Amount £1,694,091 (GBP)
Funding ID MR/S019103/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 03/2022
 
Description Wellcome Trust Programme Grant (Genetic and phenotypic characterisation of emerging virulent Clostridium difficile)
Amount £577,695 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2009 
End 05/2015
 
Description Wellcome Trust Senior Investigator award
Amount £2,100,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 04/2019
 
Description Use of NMR to study detail bacterial glycostructures 
Organisation National Research Council - Ottawa
Country Canada 
Sector Public 
PI Contribution We supplied bacterial flagellin samples that were analysed by NMR for fine detail structural characterisation
Collaborator Contribution They provided NMR data to decipher the structure of two separate flagellin glycan structures from Clostridium difficile
Impact We published two papers in J Biol Chem on the structure of the C. difficile glycosylated flagellins
Start Year 2013
 
Title THERAPEUTIC FOR TREATING CLOSTRIDIUM DIFFICILE INFECTION 
Description The invention relates to deoxyribonuclease for use in the treatment of a suspected or existing C. difficile infection; a pharmaceutical or veterinary composition or formulation comprising at least deoxyribonucleasefor use in the treatment of a suspected or existing C. difficile infection; a combination therapeutic comprising at least deoxyribonucleasefor use in the treatment of a suspected or existing C. difficile infection; a method of treating a mammal suspected of being infected with, or infected with, C. difficile comprising the use of at least deoxyribonuclease; a method of cleaning or sterilising a material or product comprising the use of at least deoxyribonuclease; and a cleaning or sterilising product impregnated with or containing at least deoxyribonuclease. 
IP Reference WO2013175172 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact Simple treatment to reduce C. difficile disease
 
Description Broadcast interviews (Newsnight, Panorama, One Show) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interest in research at LSHTM

Better understanding of science
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014
 
Description TV Broadcasts 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact BBC Breakfast TV interview on vaccine design and antimicrobial resistance
BBC one interview on Campylobacter in Food chain
One Show BBC on antimicrobial resistance
Year(s) Of Engagement Activity 2015