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 Nanoparticle Exposure Can Awaken Dormant Viruses in the Lungs
16.01.2017 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt

nachricht Cholera bacteria infect more effectively with a simple twist of shape
13.01.2017 | Princeton University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: Designing Architecture with Solar Building Envelopes

Among the general public, solar thermal energy is currently associated with dark blue, rectangular collectors on building roofs. Technologies are needed for aesthetically high quality architecture which offer the architect more room for manoeuvre when it comes to low- and plus-energy buildings. With the “ArKol” project, researchers at Fraunhofer ISE together with partners are currently developing two façade collectors for solar thermal energy generation, which permit a high degree of design flexibility: a strip collector for opaque façade sections and a solar thermal blind for transparent sections. The current state of the two developments will be presented at the BAU 2017 trade fair.

As part of the “ArKol – development of architecturally highly integrated façade collectors with heat pipes” project, Fraunhofer ISE together with its partners...

Im Focus: How to inflate a hardened concrete shell with a weight of 80 t

At TU Wien, an alternative for resource intensive formwork for the construction of concrete domes was developed. It is now used in a test dome for the Austrian Federal Railways Infrastructure (ÖBB Infrastruktur).

Concrete shells are efficient structures, but not very resource efficient. The formwork for the construction of concrete domes alone requires a high amount of...

Im Focus: Bacterial Pac Man molecule snaps at sugar

Many pathogens use certain sugar compounds from their host to help conceal themselves against the immune system. Scientists at the University of Bonn have now, in cooperation with researchers at the University of York in the United Kingdom, analyzed the dynamics of a bacterial molecule that is involved in this process. They demonstrate that the protein grabs onto the sugar molecule with a Pac Man-like chewing motion and holds it until it can be used. Their results could help design therapeutics that could make the protein poorer at grabbing and holding and hence compromise the pathogen in the host. The study has now been published in “Biophysical Journal”.

The cells of the mouth, nose and intestinal mucosa produce large quantities of a chemical called sialic acid. Many bacteria possess a special transport system...

Im Focus: Newly proposed reference datasets improve weather satellite data quality

UMD, NOAA collaboration demonstrates suitability of in-orbit datasets for weather satellite calibration

"Traffic and weather, together on the hour!" blasts your local radio station, while your smartphone knows the weather halfway across the world. A network of...

Im Focus: Repairing defects in fiber-reinforced plastics more efficiently

Fiber-reinforced plastics (FRP) are frequently used in the aeronautic and automobile industry. However, the repair of workpieces made of these composite materials is often less profitable than exchanging the part. In order to increase the lifetime of FRP parts and to make them more eco-efficient, the Laser Zentrum Hannover e.V. (LZH) and the Apodius GmbH want to combine a new measuring device for fiber layer orientation with an innovative laser-based repair process.

Defects in FRP pieces may be production or operation-related. Whether or not repair is cost-effective depends on the geometry of the defective area, the tools...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

12V, 48V, high-voltage – trends in E/E automotive architecture

10.01.2017 | Event News

2nd Conference on Non-Textual Information on 10 and 11 May 2017 in Hannover

09.01.2017 | Event News

Nothing will happen without batteries making it happen!

05.01.2017 | Event News

 
Latest News

Multiregional brain on a chip

16.01.2017 | Power and Electrical Engineering

New technology enables 5-D imaging in live animals, humans

16.01.2017 | Information Technology

Researchers develop environmentally friendly soy air filter

16.01.2017 | Power and Electrical Engineering

VideoLinks
B2B-VideoLinks
More VideoLinks >>>