Community Research and Development Information Service - CORDIS


HYPERDIFF Report Summary

Project ID: 223585
Funded under: FP7-HEALTH
Country: United Kingdom

Periodic Report Summary 3 - HYPERDIFF (The physiological basis of hypervirulence in clostridium difficile: a prerequisite for effective infection control)

Project context and objectives:

The project starting point
The inexorable rise in the incidence of the multiply-drug resistant C. difficile 027 hypervirulent strains shows no sign of abating. It is imperative that strategies to combat this and similar novel variant(s) are devised. Central to the control of this surge in incidence is both an understanding of the physiological basis of hypervirulence and an appreciation of the identity of the reservoirs of the strains driving the epidemic. At the outset of this project, little was known of the molecular basis of virulence / hypervirulence in C. difficile. The only certainty was that the two toxins (TcdA and TcdB) play a central role in disease. The participation of all other factors, known or inferred, remained conjecture.

Project objectives
The overall objective of this proposal was to determine the physiological factors that cause hypervirulence in Clostridium difficile, to provide crucial information for both the development of more informed tests for diagnosis and epidemiological studies, and the formulation of more effective countermeasures for infection control and disease management. Our basic strategy was to systematically inactivate those chromosomal genes which encode products hypothesised to be involved in pathogenesis, and to assess the effects on virulence using animal and cell culture models. The principal specific objectives were:

1. To determine any correlation between hypervirulence and toxins, by the:
- purification of toxin B (TcdB) from a non-hypervirulent and hypervirulent strain;
- generation of mutants in tcdA, tcdB and cdtAB of a non-hypervirulent and hypervirulent strain;
- determination of the relative contributions of TcdA, TcdB and CDT toxin to virulence in the in vivo model;
- identification of epitopes specific to 027 TcdB toxin, and the;
- identification of any TcdB-027 specific epitopes which are correlated to hypervirulence of C. difficile 027 strains.

2. To determine any correlation between hypervirulence and factors other than toxin, by the:
- generation of mutants in genes encoding putative colonisation / surface factors;
- generation of mutants in genes unique / divergent in 027 strains;
- measurement of the degree of attenuation of putative colonisation / surface factor mutants;
- measurement of the degree of attenuation of mutants in genes unique/ divergent in 027 genes, and;
- determination of the role of S-layer proteins in immune modulation.

3. To determine the correlation between hypervirulence and virulence factor regulation, by the:
- the establishment of the effect of antibiotic pressure on virulence factor regulation using in vivo transcriptomics;
- definitive establishment of the role of TcdC in toxin production and hypervirulence;
- determination of the role of the Agr system in virulence and hypervirulence;
- identification of any role mobile elements may play in hypervirulence;
- identification of environmental factors and regulatory genes that control virulence factor production, both in vitro and in vivo; and the
- identification of potential vaccine candidates and or therapeutic targets.

4. To determine, through epidemiological studies, the prevalence of the identified hypervirulence traits in human and animal populations, by the:
- establishment of a pan-European collection of C. difficile human strains derived from cases of both healthcare-associated disease and community-acquired disease;
- establishment of a pan-European collection of C. difficile strains of animal origin;
- comparison of the various typing systems available using the human strain collection;
- characterisation (molecular typing and colonisation properties) of the animal-derived strains; and
- refinement of available typing systems to specifically target hypervirulence markers and their subsequent use to determine the prevalence of these traits in both human and animal strains.

Objectives were pursued through eight interrelated scientific Work packages (WP2-WP9) which deployed a range of multidisciplinary approaches and technologies, including ClosTron mutant generation, transcriptome profiling, the application of various in vivo models, epitope mapping, MALDI-TOFF MS, MLVA, WAVE and epidemiology.

Project results:

HYPERDIFF objectives were pursued through eight interrelated scientific work packages (WP2-WP9) which deployed a range of multidisciplinary approaches and technologies, including ClosTron mutant generation, transcriptome profiling, the application of various in vivo models, epitope mapping, MALDI-TOFF MS, MLVA, WAVE and epidemiological approaches on both human and animal populations. Main results were:

Correlation between Hypervirulence and toxins
We re-established the importance of toxin A in Clostridium difficile infection (CDI) by showing that isogenic C. difficile mutants producing toxin A alone can cause disease in the hamster. Consequently, the development of diagnostics, vaccines and therapeutics should focus on both toxin A and B. Our data indicates that binary toxin (CDT) may also contribute to disease, and therefore hypervirulence. However, this contrasts with epidemiological data. Further research is required to determine the role of CDT but it may be prudent to test for its presence in clinical isolates through appropriate diagnostic tests. Toxin A and B genes can also be transferred between strains. This has implications for treatment plans that involve the use of non-toxinogenic strains.

Correlation between hypervirulence and factors other than Toxins
Type 027 strain R20291 adhered more strongly to epithelial cells than the non-hypervirulent strain 630, more effectively colonised mouse intestines and adhered more strongly to caeca. Inactivation of adherence genes showed considerable variation in their roles between R20291 and 630, which may contribute to virulence. Our inability to inactivate certain genes (SplA) led to the development of an assay of essentiality, and demonstrated that gldA is essential. Drugs that interfere with the activity of GldA could provide a novel treatment therapy. Compelling evidence was amassed against the current dogma, that hypervirulent / epidemic strains are more prolific in terms of spore formation. Differences are more likely in the timing of sporulation. The majority of C. difficile mobile genetic elements were shown to be transferrable. Encoded genes may allow recipient strains to rapidly exploit a new niche and cause disease.

Correlation between hypervirulence and virulence factor regulation
Transcriptomics showed that surface protein genes were differentially regulated in R20291 compared to 630 and specific genes were early up-regulated in vivo. Consequently, R20291 may possess ecological advantages that enhance GI tract colonisation. Contrary to current dogma, we show that the tcdC genotype does not affect the amount of toxin A / B produced. It seems most likely that TcdC may act like a 'safety catch' to safeguard against inappropriate toxin expression, rather than having a quantitative effect on toxin production. In view of these findings, the use of diagnostic tests based on the detection of tcdC variant sequences may not be appropriate. More important in terms of elevated toxin production, maybe a specific Agr Quorum Sensing locus which is widely distributed in hypervirulent and epidemic strains. Alternative sigma factors carried by mobile elements may also have effects on any host acquiring the element.

The prevalence and significance of hypervirulence Traits in Human and Animal Populations
A database of human and animal C. difficile isolates has been assembled and used to both develop (MALDI-TOFF) and improve (MVLA) typing systems. Our analysis has shown that assumed virulence markers, such as hyperproduction of toxins and presence of binary toxin genes, are not associated with severity and outcome of the disease in humans. C. difficile isolates obtained from human infections in the community were shown to be different to those in hospitals, but significantly resemble isolates obtained from pet and farm animals indicating that they act as a reservoir for human infection, at least in the community.

Potential Impact:

Expected final results
1) Importance of toxin A in CDI - HYPERDIFF re-established the importance of toxin A in CDI, and should encourage pharmaceutical and diagnostic companies to continue to target both toxins in developed counter measures.
2) Importance of variant TcdC alleles in hypervirulence - precise allele exchange of variant TcdC alleles established TcdC has no effect on toxin production, calling into the question the current practice of targeting TcdC variants in commercial diagnostic tests as an indicator of hypervirulence.
3) CDT binary toxin and hypervirulence - Our data suggest CDT toxin may also contribute to disease, and therefore hypervirulence. It is, therefore, prudent to test for its presence in clinical isolates.
4) Spread of C. difficile toxin potential in the GI tract - Our demonstration that the pathogenicity locus of a toxigenic strain can be transferred to non-toxigenic strains has profound implications for management of C. difficile outbreaks.
5) Relevance of S-layer proteins to hypervirulence. The demonstration that S-layer proteins induce a Th1 / Th2 response suggest they may be used as vaccine adjuvants.
6) Better measures of hypervirulence. We have shown that the genetic potential is more important than the PCR-ribotype to determine the virulence potential of a strain.
7) Identification of therapeutic targets by essentiality - We have developed a method to determine essentiality, and exemplified with CD0274. Inhibitors that interfere with the function of this enzyme would, therefore, have potential as anti-C.difficile drugs.
8) Improved molecular typing of C. difficile - Diagnostic tests directed to specific DNA inserts in hypervirulent C. difficile 027 / 078 strains were developed and patented.

Socioeconomic impact / Wider societal implications
Outputs of the project will lead to the development of new diagnostic tests and therapies, and as a consequence job creation, license fees, royalties and milestone payments:
1) Training - HYPERDIFF enabled the employment and training of a cadre of early and late stage researchers. 2) European scientific collaborations - HYPERDIFF strengthened the relations between European scientific teams fostering new collaborations, e.g. a Marie Curie ITN, CLOSTNET.
3) Engaging with civil society and policy makers -HYPERDIFF has contributed national policies regarding CDI surveillance of CDI through: (i) the development of new guidelines; (ii) reducing antibiotic prescriptions and improved hospital infection control; (iii) increasing awareness for CDI in animals and possible role of zoonotical transmission.

Dissemination activities
1) Publication in scientific journals - In total, there have been 19 peer reviewed publications (6 in preparation), 5 book chapters / conference proceedings, 14 associated papers.
2) Presentations at scientific meetings - Over 50 presentations were made by HYPERDIFF at various scientific meetings, e.g. International Clostridium difficile Symposia (2010), international conference on the molecular biology and pathogenesis of the Clostridia (2009 and 2012) and European society of clinical microbiology and infectious diseases (2000, 2010 & 2011). 3) Public engagement and media activities - All of the partners took part in media interviews (TV, radio and newspapers), open days, school visits and special public sessions at international meetings.

Exploitation of results
HYPERDIFF partners have been contacted by various commercial enterprises concerned with diagnosis or therapy as a result of their outputs. These include, companies seeking to develop new diagnostic tests, companies developing vaccines and therapeutics. Patents covering gene tools have been licensed by several companies for exploitation in other fields (industrial biotechnology). Additional funding from national bodies has also been awarded. An international collection of animal strains curated by the Institute of Public Health Maribor has also been established.

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Paul CARTLEDGE, (Head of Research Grants and Contracts)
Tel.: +44-115-9515679
Fax: +44-115-9513633
Record Number: 53838 / Last updated on: 2012-10-22
Information source: SESAM
Collaboration sought: N/A