Global assignment of the function of Salmonella genes in livestock
Lead Research Organisation:
University of Cambridge
Department Name: Veterinary Medicine
Abstract
Bacteria of the species Salmonella enterica are a threat to public health and sustainable agriculture. Over 2460 distinct variants of the organism have been described and these can be divided into two groups; serovars restricted or adapted to a given host that cause systemic illness (e.g. S. Typhi, which causes Typhoid fever in humans) and serovars causing gastroenteritis in a wide range of hosts (e.g. S. Typhimurium). Typhoid fever is a largely water-borne disease perpetuated by unsanitary conditions and 21 million human illnesses and 216,510 deaths are estimated to occur each year worldwide. Gastroenteritis caused by non-typhoidal Salmonella strains is also common, with an estimated 1.4 million cases and 600 deaths per annum in the United States alone. These infections are often associated with the consumption of foods derived from chickens, pigs and cattle owing to the ability of the bacteria to colonise the intestines of such animals. Strategies to reduce the carriage of S. enterica in food-producing animals are expected to lower the incidence of human infections and improve animal health. Whilst vaccines exist for the control of Salmonella in poultry, they confer weak and variable protection against different serovars and it is widely agreed that effective vaccines or treatments are needed for use in other livestock species. Furthermore, not all S. enterica serovars found in animals pose a threat to humans and tools are needed to predict the epidemic potential of such strains. The development of novel vaccines, treatments and diagnostics for Salmonella requires an understanding of the molecular events underlying intestinal colonisation. Our laboratories have identified some S. Typhimurium factors needed for colonisation of chickens, pigs and calves, but the function of less than one fifth of all Salmonella genes has so far been examined. S. Typhimurium infections in calves and pigs result in acute gastroenteritis whilst carriage in chickens tends to be asymptomatic. The reasons why Salmonella causes disease in one host but not another are not understood. Furthermore, it is not known why some S. enterica serovars cause systemic disease in a given host whereas others are restricted to the alimentary tract. We have invented a new method that permits the simultaneous screening of thousands of individual Salmonella mutants, each lacking a particular character, during infection of animals. The method (transposon-mediated differential hybridisation, TMDH) can rapidly describe the role of virtually all Salmonella genes during infection and relies on detecting signals from each mutant on a chip on which all the bacterial genes are arrayed. Bacterial genes are mutated at random then the mutants are assembled into large pools that can be inoculated into animals. The use of large pools dramatically reduces the number of animals that are needed. If a mutant is represented in the inoculum, but not in a pool recovered after the bacteria have passed through the animal, it can be inferred that the character disrupted in that mutant is important for colonisation. TMDH has been validated in mice, but it now needs to be used in food-producing animals. The method supersedes existing approaches, since it can inform the researcher when a gene is, or is not, required during infection without the need to isolate and examine the mutated gene. We propose to screen 10,000 S. Typhimurium mutants by this method for their ability to colonise the intestines of chickens, pigs and cattle. This will provide insights that cannot be obtained using surrogate rodent or cell-based assays and relies on the unique expertise and facilities at the applicants' laboratories. TMDH will greatly accelerate the identification of factors needed for Salmonella pathogenesis, some of which may be suitable as targets for drugs or included in a vaccine. Treatments developed for use in livestock may also be suitable for use in humans against typhoid fever.
Technical Summary
The molecular basis of intestinal colonisation, induction of enteritis and systemic translocation by Salmonella is incompletely understood. Human infections are frequently acquired via consumption of products derived from poultry, pigs and cattle and effective vaccines or treatments for control of Salmonella in food-producing animals are lacking. We have invented a powerful new method to study the function of virtually all transposable genes in vivo. Transposon-mediated differential hybridisation (TMDH) uses Tn5 and Mu-based transposons containing T7 and SP6 promoters enabling the composition of mutant pools and location of insertion sites to be simultaneously determined by hybridisation of run-off transcripts to high-density oligonucleotide arrays. The method has been successfully used to screen 10,000 S. Typhimurium mutants in a murine typhoid model, providing insights into the role of every mutated gene during infection. The method supersedes existing approaches since it informs the researcher when a gene is, or is not, required during infection without the need for cloning and sequencing of Tn-ends and allows many more mutants to be screened. Screening of signature-tagged mutants at IAH and Cambridge has indicated that S. Typhimurium uses both conserved and host-specific factors to colonise the intestines of chickens, pigs and cattle. However, the function of less than a fifth of the Salmonella genome has been probed. Furthermore, analysis of mutants in streptomycin-pretreated mice has indicated that the mouse is often incapable of detecting factors needed in livestock species. Having validated TMDH in mice, we propose to screen the S. Typhimurium mutant libraries in chickens, pigs and calves. These screens will be the most relevant and exhaustive yet described and will enable us to ascribe an attenuation index for every transposed gene in each host. The role of selected genes will be confirmed by construction and characterisation of defined mutants.
Organisations
Publications
Carnell SC
(2007)
Role in virulence and protective efficacy in pigs of Salmonella enterica serovar Typhimurium secreted components identified by signature-tagged mutagenesis.
in Microbiology (Reading, England)
Chaudhuri RR
(2009)
Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH).
in BMC genomics
Chaudhuri RR
(2013)
Comprehensive assignment of roles for Salmonella typhimurium genes in intestinal colonization of food-producing animals.
in PLoS genetics
Eckert SE
(2011)
Retrospective application of transposon-directed insertion site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in cattle.
in Journal of bacteriology
Langridge G
(2009)
Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants
in Genome Research
M Stevens (Author)
(2009)
Genetic analysis of Salmonella and E. coli pathogenesis in food-producing animals.
M Stevens (Author)
(2007)
Toward control of enteric bacterial diseases using novel 3R methods
M Stevens (Author)
(2008)
Toward ctonrol of enteric bacterial infections of using novel 3R methods
M Stevens (Author)
(2009)
Molecular insights into Salmonella pathogenesis in food-producing animals
Description | 1) A library of 8550 S. Typhimurium strain SL4/74 transposon mutants was created and screened by oral inoculation of chickens, calves and pigs, following optimization of pool complexity and the timing and site of recovery. Mutants were reliably negatively-selected across replicates and pools. 2) Insertion sites and attenuation scores were assigned for c. 6600 S. Typhimurium mutants screened in chickens, calves and pigs, revealing the role of c. 3200 different genes in intestinal colonization. |
Exploitation Route | This provides genomic scale information about genes required to infect/colonise livestock with Salmonella Typhimurium. It is information that could be the basis of a number of vaccine routes to prevent infection with this organism, if the economic climate for this kind of vaccine were better. |
Sectors | Agriculture Food and Drink Healthcare Pharmaceuticals and Medical Biotechnology |
Description | This work established which genes in Salmonella Typhimurium are responsible for infection and host adaptation by infecting livestock species (calves, pigs and chickens) with large pools of transposon mutants and assigning fitness scores to mutants using TMDH and TraDIS. The technology has been applied to the study of other pathogens in other natural hosts or animal models. The genes identified could be used as novel vaccine or antimicrobial targets. |
First Year Of Impact | 2009 |
Sector | Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | A multivalent vaccine and single platform diagnostic for bacterial respiratory diseases of pigs |
Amount | £2,303,325 (GBP) |
Funding ID | BB/G019274/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2009 |
End | 11/2015 |
Title | Transposon (Tn 5 and Mu) mutant libraries of Salmonella Typhimurium SL1344 and a Tn5 mutant library of ST4/74 |
Description | 5,000 Tn5 mutants and 5,000 Mu mutants of S. Typhimurium SL1344 stored as glycerol stocks of individual mutants in 96-well plates. 10,000 Tn5 mutants of S. Typhimurium ST4/74 stored as glycerol stocks of individual mutants in 96-well plates. |
Type Of Material | Biological samples |
Year Produced | 2007 |
Provided To Others? | Yes |
Impact | These mutants have been given to several different reseacrh groups, for example, a PhD student at the Royal Free Hospital used some of these mutants as part of his PhD studies, PhD awarded, 2011. |
URL | http://www.ncbi.nlm.nih.gov/pubmed/19649318 |
Title | in vivo TraDIS (TMDH) |
Description | This research method involves making large libraries of transposon mutants in bacteria and using them in large pools to infect an animal or ex vivo model of infection. Transposon mutants are recovered following infection and identified by next-generation sequencing (or DNA micro-arrays), using novel bioinformatic software. Any mutants with reduced fitness in comparison with the majority of the mutants in the pool can be identified and the mutations can be incorporated in to live-attenuated vaccine candidate strains. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2009 |
Provided To Others? | Yes |
Impact | This has been used to investigate the pathogenesis of several different bacteria (including Salmonella Typhimurium, Salmonella Typhi, Streptococcus suis, Haemophilus parasuis), generating a large amount of data about which genes are required for infection and the genes that are not required for infection. This has also identified new mutations for use in live-attenuated vaccine strains and surface proteins that are candidates for sub-unit vaccines. Several high-impact research papers have been published that describe the use of this research method. |
URL | http://www.ncbi.nlm.nih.gov/pubmed/19649318 |
Title | Salmonella TraDIS website |
Description | This website allows the user to search the results of in vivo TraDIS experiments which used pools of transposon mutants to infect mice, calves, pigs and chickens during the course of two BBSRC funded grants (APG19115 and BB/D017947/1). Transposons insertions are mapped to the Salmonella genome sequence and the fitness of each mutant during infection of the four different hosts can be viewed. Genes of interest can be selected to view on xBASE to give further details on their products and functions. |
Type Of Material | Database/Collection of data |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | This website gives a huge amount of data on genes required by Salmonella for infection of livestock species and mice, and also shows which genes are not required for infection. This will be useful for identifying vaccine candidates and novel targets for anti-microbials. |
URL | http://lin5.shef.ac.uk/dalliance/ |
Title | Analysis of TraDIS data |
Description | Method for analysis of TraDIS data, applicable to any bacterial transposition and a webtool for viewing the data from experiments with transposon libraries libraries in Salmonella screened in vivo in mice (2009) and livestock species (2013). |
Type Of Technology | Webtool/Application |
Year Produced | 2009 |
Impact | Other researchers have been able to view the data. |
URL | http://www.ncbi.nlm.nih.gov/pubmed/23637626 |