Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

TraDIS technique tackles typhoid

20.10.2009
First high-throughput analysis of every Salmonella Typhi gene

For the first time, researchers are able to look at the need for every gene in a bacterial cell in a single experiment. The new method will transform the study of gene activity and the search for weaknesses in bacterial armouries.

Using a newly developed, next-gen sequencing method, a team established which genes Salmonella Typhi needs to survive and which are more of a luxury. The results and the method will be a boon to scientists tackling bacterial disease, allowing them to capitalize on the abundance of genomic sequence data from next-generation sequencing technologies.

Every year 22 million people are infected and 220,000 die from infection with S. Typhi. It is a special threat in the developing world, in areas with poor sanitation or a lack of clean drinking water.

The team were able to look at almost all the genes in S. Typhi and showed that it needs only 356 genes for survival: 4162 genes were not essential. Knowing which genes are essential to the survival of pathogens, researchers can seek treatments to target those genes.

"We developed a new method that is ten times more powerful than any previous technique," says Sanger Institute graduate student Gemma Langridge, one of the first authors on the paper. "By combining transposon-induced mutagenesis – a method whereby small chunks of cut-and-paste DNA sequence are inserted into the genome effectively disabling individual genes – and high-throughput sequencing, we have been able to determine which genes are essential for the survival of S. Typhi and which are non-essential."

"Crucially, our new method allows us to achieve all this in just a single experiment."

Using the novel method, which the team have named TraDIS (Transposon Directed Insertion site Sequencing), they inserted transposons into the S. Typhi genome to generate more than one million mutants. They then grew the bacteria and used next-generation sequencing to directly identify 370,000 insertion sites in the S. Typhi genome – an average of more than 80 insertion sites per gene. Previous methods produce only a few mutations per gene.

If a transposon inserts into an essential gene, the gene is silenced and that mutant cell will not grow and it – and the transposon insert – will be absent from the mutant pool. By sequencing DNA from the entire pool – approximately 1 million mutants in total – the team were able to identify genes in which no transposon insertions had been detected.

In a single experiment using the TraDIS method, the team were able to determine whether or not 99.6% of the S. Typhi genes are essential to its survival.

"Sequencing centres such as ours can produce vast amounts of genomic data at a pace unimaginable just a few years ago," explains Professor Julian Parkhill, Director of Sequencing and head of Pathogen Genomics at the Sanger Institute. "One of our aims is to develop high-throughput research methods that can exploit this explosion of genetic data, to ensure these resources can be used effectively. We can now discover which of all the genes in an organism are essential to its survival or required for growth under special conditions, such as infection. Our new TraDIS method will make a dramatic difference to the ability to carry out such genome-wide research."

Importantly, the team applied the method to a clinical problem by looking at how S. Typhi might survive in humans. Typhoid can be spread by carriers who, without showing symptoms, act as reservoirs, storing the bacterium in the gallbladder and passing it to others. The most famous such carrier was Typhoid Mary, who worked in the food industry in the US and spread typhoid fever without exhibiting any symptoms herself.

But, bacteria cannot survive in the fairly hostile environment of the gall bladder unless they are tolerant to bile – the fatty fluid secreted by the gall bladder. Looking at genes involved in bile resistance, allows us to see which genes are essential for helping S. Typhi persist in a carrier.

"We grew the bacteria in ox bile to pick out genes required for bile tolerance," says Keith Turner, Sanger Institute investigator and a senior author on the paper. "We found 169 genes involved in bile tolerance – many of these had not been suspected before and more than 30 are genes not characterized at all.

"Using TraDIS, we have highlighted several possible new targets for treatment that would pick on S. Typhi's need to survive in the gall bladder."

For the first time, it is possible to paint a comprehensive picture of essential, advantageous or burdensome genes in many phases of the bacterial life cycle, to determine functions necessary to support them throughout their entire disease cycle. Such a picture is important for discovery of new targets for treatment.

This elegant new method exemplifies how high-throughput research allows scientists to determine systematically the function of or requirement for individual genes in a single experiment, opening the door for similar analyses of other pathogenic genomes in the future.

Publication Details
Langridge G C, Phan M-D, Turner D J et al. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Research

Published online before print at doi: 10.1101/gr.097097.109

Funding

This work was supported by the Wellcome Trust.

Participating Centres

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
Environmental Research Institute, University College, Lee Road, Cork, Ireland
Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, UK
Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK

Laboratory of Gastrointestinal Pathogens, Centre for Infections, Health Protection Agency, Colindale, London, UK

The Wellcome Trust Sanger Institute, which receives the majority of its funding from the Wellcome Trust, was founded in 1992. The Institute is responsible for the completion of the sequence of approximately one-third of the human genome as well as genomes of model organisms and more than 90 pathogen genomes. In October 2006, new funding was awarded by the Wellcome Trust to exploit the wealth of genome data now available to answer important questions about health and disease. http://www.sanger.ac.uk

The Wellcome Trust is the largest charity in the UK. It funds innovative biomedical research, in the UK and internationally, spending over £600 million each year to support the brightest scientists with the best ideas. The Wellcome Trust supports public debate about biomedical research and its impact on health and wellbeing. http://www.wellcome.ac.uk

Don Powell | EurekAlert!
Further information:
http://www.sanger.ac.uk

More articles from Life Sciences:

nachricht Cryo-electron microscopy achieves unprecedented resolution using new computational methods
24.03.2017 | DOE/Lawrence Berkeley National Laboratory

nachricht How cheetahs stay fit and healthy
24.03.2017 | Forschungsverbund Berlin e.V.

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Giant Magnetic Fields in the Universe

Astronomers from Bonn and Tautenburg in Thuringia (Germany) used the 100-m radio telescope at Effelsberg to observe several galaxy clusters. At the edges of these large accumulations of dark matter, stellar systems (galaxies), hot gas, and charged particles, they found magnetic fields that are exceptionally ordered over distances of many million light years. This makes them the most extended magnetic fields in the universe known so far.

The results will be published on March 22 in the journal „Astronomy & Astrophysics“.

Galaxy clusters are the largest gravitationally bound structures in the universe. With a typical extent of about 10 million light years, i.e. 100 times the...

Im Focus: Tracing down linear ubiquitination

Researchers at the Goethe University Frankfurt, together with partners from the University of Tübingen in Germany and Queen Mary University as well as Francis Crick Institute from London (UK) have developed a novel technology to decipher the secret ubiquitin code.

Ubiquitin is a small protein that can be linked to other cellular proteins, thereby controlling and modulating their functions. The attachment occurs in many...

Im Focus: Perovskite edges can be tuned for optoelectronic performance

Layered 2D material improves efficiency for solar cells and LEDs

In the eternal search for next generation high-efficiency solar cells and LEDs, scientists at Los Alamos National Laboratory and their partners are creating...

Im Focus: Polymer-coated silicon nanosheets as alternative to graphene: A perfect team for nanoelectronics

Silicon nanosheets are thin, two-dimensional layers with exceptional optoelectronic properties very similar to those of graphene. Albeit, the nanosheets are less stable. Now researchers at the Technical University of Munich (TUM) have, for the first time ever, produced a composite material combining silicon nanosheets and a polymer that is both UV-resistant and easy to process. This brings the scientists a significant step closer to industrial applications like flexible displays and photosensors.

Silicon nanosheets are thin, two-dimensional layers with exceptional optoelectronic properties very similar to those of graphene. Albeit, the nanosheets are...

Im Focus: Researchers Imitate Molecular Crowding in Cells

Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to simulate these confined natural conditions in artificial vesicles for the first time. As reported in the academic journal Small, the results are offering better insight into the development of nanoreactors and artificial organelles.

Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

International Land Use Symposium ILUS 2017: Call for Abstracts and Registration open

20.03.2017 | Event News

CONNECT 2017: International congress on connective tissue

14.03.2017 | Event News

ICTM Conference: Turbine Construction between Big Data and Additive Manufacturing

07.03.2017 | Event News

 
Latest News

Argon is not the 'dope' for metallic hydrogen

24.03.2017 | Materials Sciences

Astronomers find unexpected, dust-obscured star formation in distant galaxy

24.03.2017 | Physics and Astronomy

Gravitational wave kicks monster black hole out of galactic core

24.03.2017 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>