Research within the Salmonella Group at the IFR fits into the UK’s grand challenge of maintaining global food security into the 21st century by focusing on:
The regulation of S. Typhimurium virulence gene expression in response to infection
One of the research areas of the IFR Salmonella group is to investigate the mechanisms by which S. Typhimurium virulence genes are controlled. We have been addressing this objective using a next generation transcriptomic approach using differential RNA-seq; (dRNA-seq). The dRNA-seq approach enriches for primary, rather than processed transcripts; this enables the precise identification of transcriptional start sites for genes and non-coding RNAs (ncRNAs) on a genome-wide scale 1,2.
We used dRNA-seq to define the complete (primary) transcriptome of S. Typhimurium during growth conditions which specifically induce Salmonella Pathogenicity Island 1 (SPI1) expression 1. The latter publication describes the first extensive and accurate map that shows the precise nucleotide positions of the transcriptional start sites (TSSs) for 78% of the S. Typhimurium genome under SPI1 inducing conditions. The precise location of the TSSs enabled the prediction of the structure of 625 operons and 169 alternative sigma factor binding sites. We also discovered extensive transcription of ncRNAs’ including 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). More recently we have used dRNA-seq to define the transcriptomic architecture for S. Typhimurium under conditions in which genes required for expression of SPI2 are expressed 2. The SPI1 cluster encodes genes required for the invasion of S. Typhimurium into host cells whereas SPI2 encodes genes required for replication and survival of S. Typhimurium within host cells. Characterising the transcriptional architecture of S. Typhimurium under these conditions has resulted in the discovery of potential new control mechanisms used to regulate virulence genes expression. In addition, the above publications define further the role of the bacterial signal molecule, guanosine tetraphosphate (ppGpp) in controlling nearly all virulence gene expression in S. Typhimurium (see below).
The precise locations of all of the S. Typhimurium TSSs (genes and ncRNAs) are described in the supplementary data in our publications 1,2. The mapped dRNA-seq reads from the latter publications can also be viewed in the embedded genome browser JBrowse (http://jbrowse.org/) at the following site: http://opendata.ifr.ac.uk/salseq.
The role of global gene regulators in bacterial virulence
Guanosine tetraphosphate (ppGpp) is a bacterial signal molecule that modulates S. Typhimurium virulence gene expression in response to environmental stresses 2. We have previously shown that ppGpp is required for the expression of nearly all of the genes associated with virulence in S. Typhimurium 3 and it has also been shown that ppGpp plays a prominent role in the virulence of many other bacterial pathogens. To gain further insights into the global role that ppGpp plays in regulating virulence gene expression we used dRNA-seq to compare the transcriptome of wild-type S. Typhimurium with that of a ppGpp null mutant strain 1,2. We found that approximately a third of coding and non-coding RNA transcription was ppGpp-dependent under growth conditions where SPI1 and SPI2 were expressed, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment 1,2.
The metabolism of S. Typhimurium during infection
A long-standing microbiological question is how the metabolic pathways of Salmonella (and those of other intracellular bacterial pathogens) adapt to enable the bacterium to survive and replicate within host cells such as macrophages, which normally kill pathogenic organisms. From a previous BBSRC research grant we showed for the first time which nutrients and metabolic pathways S. Typhimurium uses to enable survival in macrophages 4,5. S. Typhimurium relies on glucose to the exclusion of almost any other carbon source for replication in macrophages and requires glycolysis for survival. This exciting and innovative work has continued with a currently active BBSRC grant to determine the relationship between Salmonella energy metabolism and virulence during infection of epithelial cells and macrophages.
Novel methods for the inactivation of Salmonella
Traditional means to control food spoilage and microbiological safety hazards, such as sterilisation, curing or freezing are not compatible with consumer demands for fresh produce. Therefore research efforts are directed towards developing alternative robust antimicrobial processes suitable for application to minimally processed foods. One such process is the use of non-thermal ionized gases (cold gas plasma; CAP). For CAP to be successfully adopted by the food industry, factors which affect its potential for microbial inactivation must be evaluated. We reviewed the application of CAP for inactivating Salmonella 6 and have started to characterise some of the factors which are involved 7.
1. Ramachandran V. K., Shearer N., Jacob J., Sharma C. M., Thompson A. (2012) The architecture and ppGpp dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression. BMC Genomics. 13:25.
2. Ramachandran V. K., Shearer N., Thompson A. (2014) The primary transcriptome of Salmonella enterica serovar Typhimurium and its dependence on ppGpp during late stationary phase. PLoS ONE (In Press).
3. Thompson A. , Rolfe M. D., Lucchini S., Schwerk P., Hinton J.C.D., Tedin K. (2006) The bacterial signal molecule, ppGpp, mediates the environmental regulation of both the invasion and intracellular virulence gene programs of Salmonella. Journal of Biological Chemistry 281:30112.
4. Bowden S.D, Rowley G., Hinton J.C.D., Thompson, A. (2009) Glucose and glycolysis are required for the successful infection of macrophages and mice by Salmonella enterica serovar Typhimurium. Infect. & Immun. 77:3117.
5. Bowden S.D, Ramachandran V.K., Knudsen G.M., Hinton J.C., Thompson, A. (2010) An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages. PLoS One. 5(11):e13871.
6. Fernández, A. and Thompson, A (2012) The inactivation of Salmonella by cold atmospheric plasma treatment. Food Research International. 45(2):678
7. Fernández Rodriguez A. F. R., Shearer N. S., Wilson D. R. W., Thompson A. (2011) Effect of microbial loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella enterica serovar Typhimurium. International Journal of Food Microbiology 152: 175.
I was awarded a BSc in Biochemistry from the University of Kent, Canterbury and a PhD in Molecular Biology from UMIST, Manchester. I joined the IFR in 1996 and jointly set up and headed the IFR microarray facility in 2000. I became leader of the IFR Salmonella group in 2009. My research interests include the role of signal transduction molecules and pathways in the regulation of Salmonella virulence gene expression as well as how Salmonella metabolism adapts during the infection process. Over the previous five years I have been awarded two BBSRC funded research grants and filed international patents for developing live attenuated Salmonella vaccines.
I was awarded a BSc in Biological Sciences in 2007 and a PhD in 2012 by the University of Birmingham, UK. For my PhD thesis, I studied the electron transfer chains of the obligate human pathogen, Neisseria gonorrhoeae. I joined the IFR as a Postdoctoral Training Fellow in February 2012, where my current research focuses on the intracellular energy metabolism of Salmonella Typhimurium. I am interested in how the bacterium’s localisation within either epithelial cells or macrophages affects which metabolic pathways it uses to conserve energy and protect itself from host defence mechanisms, such as oxidative and nitrosative bursts. My research involves the use of cutting edge proteomic techniques to quantify changes in the abundance of particular proteins within Salmonella isolated from infected mammalian cells.
I was awarded my degree in Microbiology from the University of East Anglia in 1996, and a PhD in 2000. For my PhD thesis I studied the regulation of denitrification in Paracoccus denitrificans. Subsequent postdoctoral positions at UEA and JIC focused on nitric oxide signalling in Paracoccus denitrificans, regulation of nitrogen fixation in Azotobacter vilnlandii and the purification of plant and microbial signalling proteins. I joined the IFR in 2006 and began work in the Campylobacter group on host acute stress responses and the regulation of Campylobacter jejuni virulence factors. I was awarded an IFR-funded competitive research scholarship in 2008 and won a joint IFR/UEA synergy award in 2009 to study the role of signalling molecules in Campylobacter. In 2010 I joined the IFR Salmonella group where my research interest in the role of signalling molecules and transduction pathways in relation to virulence gene expression continues.
Dr. Ana Fernández Rodríguez
Dr. Vinoy Ramachandran