Understanding and exploiting regulation in pathogenic enteroaggregative Escherichia coli

Many bacteria cause diarrhoea in humans because, when they are ingested, they colonise the lower gut by attaching to the lining of the intestine. They then produce products that cause abnormal levels of fluid build-up in the host's gut. For most people, such diarrhoea is merely an irritation, but, for some, it can be life-threatening. Currently, one especially troublesome cause of diarrhoea, in both developed and developing countries, is enteroaggregative Escherichia coli (EAEC). This harmful organism probably evolved from harmless Escherichia coli strains following the acquisition of a score or so of extra genes that allow it to attach to human intestines and cause diarrhoea. Studies with EAEC and with other harmful bacteria have shown that the expression of these extra genes, which cause disease, is tightly controlled. For EAEC, and related harmful bacteria, we now know what the principal regulators are. Interestingly, the most important of these regulators was acquired with the other extra genes, and its role appears to be mainly to make sure that the harmful bacteria only express the disease-causing genes when the harmful bacteria are in the host.

Hence the main aims of the work proposed here are to do experiments that will tell us how the main regulator is activated when the bacteria get to the gut, and how the activity of the regulator is turned on and turned off. We plan to look at EAEC strains from patients from different countries to see if the same mechanism is always used and also to check the harmful genes in the different strains. Once we have understood how the regulator works, we will use genetic engineering techniques to make derivatives of the regulator that are defective and so will interfere with virulent strains. We will devise ways to deliver the defective regulator to harmful cells during infection with a view to developing a new way to treat bactreial infection that involves 'disarming' the harmful bacteria rather than killing them.

Enteroaggregative Escherichia coli (EAEC) is an important human pathogen that is responsible for causing diarrhoea in both adults and children in industrialised and developing countries. Infection proceeds by colonisation of the human intestinal mucosa and this involves the expression of various gene products that are encoded by a plasmid, which can be regarded as mobile vehicle of virulence. Previous studies have shown that the expression of plasmid-encoded virulence genes is tightly regulated at the level of transcript initiation, and the 'master' regulator, which is also plasmid-encoded, is AggR, a transcription activator. The activity of AggR is controlled by a small anti-activator protein but the precise nature of the inducing signal is not yet clear. We propose to create libraries of AggR and Aar mutants and, working with the paradgm 042 EAEC strain, we will screen for derivatives of Aar that 'jam' AggR in the non-induced state and derivatives of AggR that recognise binding targets normally but are unable to activate virulence gene expression. Genes encoding these mutant forms of Aar and AggR will be transferred to plasmids and bacteriphage, which will be developed to suppres EAEC infections. The properties of the mutants will be studied in order to understand the details of the Aar-AggR interaction and how virulence is induced. In the second part of the project, we will use proteomics to catalogue the proteins that control the EAEC strain 042 virulence plasmid, and measure the changes that occur during and after the induction of virulence. In parallel, we will compare the organisation of virulence genes in EAEC strains collected from patients in Rio de Janeiro and Assiut in Egypt.

The proposed project, which is highly multidisciplinary, will impact the research community, industry and wider public in many ways:

Impact on the research community: As highlighted above, we will address the issue of how the expression of the pathogenicity determinants of harmful bacteria are triggered. This will inform and impact upon researchers interested in bacterial physiology, gene expression, host-pathogen interactions and biofilm formation.

Impact on the training and education of future scientists: The project will directly contribute to the training of early career researchers through associated students (at graduate and undergraduate level) who will benefit from cross-disciplinary training at the intersection of microbiology, host-pathogen interactions, chemical engineering and biophysics. Students are usually very excited to work on practical lab projects focusing on host-pathogen interactions with us, and especially value our laboratory's direct approaches to understanding microbial processes. We will also use elements of the project to introduce school students to current research through the annual IMI Summer School. This will broaden the impact of the project on training the next generation of future scientists.

Impact on industry and public health: Through this project we will understand how bacteria perceive host tissue to trigger their virulence program. This will lead to reagents that can be used to combat infection. Novel ways to treat EAEC infections in humans are highly sought after at the moment, due to growing problems with resistance to antimicrobial agents. In addition, antibiotics are often counter-indicated in infections as they can trigger the production of shiga toxins, which lead to severe complications such as hemolytic uremic syndrome and increased morbidity and mortality. Our research will ultimately help to replenish the drug development pipeline with novel molecules, which will alleviate some of the problems we have encountered with antibiotics. This will be a direct impact and is achievable.

In addition, longer term, the work will directly inform other ways in which we can interfere with bacterial virulence, for example by devising molecular approaches that block either host attachment or signal transduction, in order to disrupt virulence gene expression. We have seen in the past that the use of antibiotics to stop colonization of food animals with food-borne pathogens has only aggravated our problems with antibiotic resistance. Thus, novel ways to stop colonization with bacterial pathogens will be of immense value to the agri-food industry and help ensure our supply with safe and healthy food in the future. Our research will also inform the design of smart antimicrobial surfaces, which will be useful in places where environmental bacterial persistence or biofilm formation is a problem (e.g. hospitals, food industry, waste water treatment plants).