Quorum sensing and lifestyle switching in Yersinia.

The Yersinia are bacteria which occupy a prominent place in the history of mankind and microbiology. Yersinia pestis, the causative agent of bubonic and pneumonic plague, has claimed millions of lives in periodic pandemics, influencing human history possibly to a greater extent than any other bacterium. The Yersiniae also include species which are pathogenic for animals (farmed and wild), birds and fish. For example, Y. pseudotuberculosis whose lifestyle alternates between the food/water environment and the mammalian gastrointestinal tract, infects livestock as well as captive zoo animals and birds. In humans it causes gastro-intestinal infections and 'far east scarlet-like' fever which involves a severe toxic shock syndrome. Y. pestis evolved from Y. pseudotuberculosis around 20,000 years ago and although these pathogens are >98% identical at the genetic level they cause very different diseases. However the Yersinia species which cause human infections all possess the same pYV extra-chromosomal plasmid which is essential for virulence since it enables Yersinia to subvert its host immune response. As unicellular micro-organisms, the Yersiniae are capable of adapting to diverse environmental stresses (e.g. widely fluctuating temperatures) that facilitate survival in, and migration from, soil and water environments into different hosts (both insects and animals). Although bacteria are single-celled, they can co-ordinate their behaviour by communicating via chemical signal molecules and by forming surface-associated communities known as biofilms. Here, bacteria become enmeshed in a 'slime' layer which confers protection in extreme environments and from the immune system and antibiotics. With respect to Yersinia, bubonic plague is transmitted by fleas whose feeding is blocked by a dense biofilm of Y. pestis in their digestive tracts. Y. pestis also blocks the feeding of the nematode worm, Caenorhabditis elegans, by forming a biofilm around its head. Some Y. pseudotuberculosis strains also readily form biofilms on C. elegans and because it is less dangerous as a pathogen than Y. pestis, it offers a much safer and simpler means of investigating biofilm development on living tissues. This biofilm model is also attractive because it is difficult to study biofilms in the mammalian host. C. elegans shares many genes with humans and so the C. elegans/Yersinia model can be used to identify in vivo genetic features of both the pathogen and the host that contribute to biofilm-mediated interactions which have interesting implications for both the Yersinia/flea and human biofilm-centred infections. We discovered that Y. pseudotuberculosis uses a sophisticated two channel quorum sensing system to make lifestyle decisions according to the prevailing local environmental conditions which help the organism decide whether to build a biofilm, become cytotoxic by secreting Yop proteins or swim away and find a new niche to colonize. This research project aims to gain insight, at the molecular level, into the signalling cascade used by Y. pseudotuberculosis to make these lifestyle decisions including whether to retain the pYV virulence plasmid. We will also seek to gain further insights into the role of the C. elegans host during biofilm formation: the surface ligands to which Yersinia attaches; and, the signalling processes occurring during development of the biofilm. This work will not only inform us about the basic biology of disease causing bacteria but in the longer term may help us to identify novel targets for the prevention or treatment of disease in humans and other animals. This is especially important with respect biofilms which are often the cause of chronic infections. These are very difficult to eradicate and therefore investigating how biofilms develop on living tissues may uncover novel ways for their disruption and prevention.

Yersinia pseudotuberculosis is an enteric pathogen that has a lifestyle which alternates between the food/water environment and the mammalian gastrointestinal tract. Its virulence depends on the pYV plasmid which harbours the yop virulon, a type III secretion (T3S) system required for subversion of host defences. Some Y.pseudotuberculosis strains form biofilms on the anterior cuticle of Caenorhabditis elegans blocking feeding. This offers a simple means of investigating biofilm development in vivo on a living surface which has the major benefit that both bacterial and host responses can be studied. Previously we discovered that Y. pseudotuberculosis employs quorum sensing (QS) to make lifestyle decisions with respect to motility, T3S, pYV plasmid maintenance, metabolism and biofilm formation on C. elegans. In addition, QS itself appears to be regulated via N-acetylglucosamine (GlcNAc) and we have identified several key C. elegans genes differentially regulated in response to Yersinia biofilms. The present project aims to obtain detailed molecular insights into the yersinia signalling pathways involved and the functions of the C. elegans genes differentially regulated in response to biofilms. We will:(1) establish whether GlcNAc is the master metabolic signal controlling QS; (2) determine how QS controls the hut operons; (3) investigate whether HutC regulates motility, T3S and biofilm formation via FlhDC; (4) elucidate whether QS regulates virF and the yop virulon directly or indirectly via the HutC/FlhDC pathway; (5) establish how QS maintains the pYV plasmid; (6) determine the relationship between suspended and C. elegans biofilms; (7) identify the C. elegans ligands that promote biofilm formation; and, (8) investigate how C. elegans responds to Yersina biofilms. This research provides a unique opportunity to study the inter-relationship of several important fundamental bacterial processes as well as providing new information in host/invertebrate interactions.

This research project is directed towards the acquisition of new molecular insights into the regulatory networks employed by pathogenic bacteria to make social, lifestyle decisions particularly in the context of virulence, host-pathogen interactions and biofilm formation. It has become apparent that most bacterial infections involve biofilm formation on host tissues. This is especially important since biofilms cause chronic infections and are highly refractory to conventional antibiotics. To date, no simple in vivo model for biofilms in living tissues has emerged and the Yersinia/C. elegans system is the first such model to offer a means to identify the genetic features of both pathogen and host that contribute to biofilm-mediated interactions. It will generate fundamental information which facilitates target identification and interventions for testing with significant downstream commercial implications. It will also contribute to the 3Rs by providing a simple cost effective in vivo model amenable to high-throughput screening for novel antibiofilm agents. The data generated will be of interest to a broad group of biological researchers in both academia and industry. The project specifically addresses the BBSRC research priority to improve global security which is impacted on by infectious diseases that are responsible for ~13 million deaths worldwide and over 25% of the annual mortality. The increasing incidence of antibiotic resistance in pathogenic bacteria allied with the dearth of newly developed antibiotics poses a further major threat to human and animal healthcare. This aspect of the work may potentially have a positive social impact by improving the health of the nation via the development of new prophylactic or therapeutic agents. This has a positive outcome for the economy by reducing the proportion of the workforce unable to work as a consequence of contracting infection. The project will generate a number of scientific tools and resources including strains, mutants, and reporter gene fusions as well as a significant amount of transcriptome data, all of which will be made available to the research community. We place strong emphasis on the early exploitation of research and our groups have been successful at doing this. Exploitation of our research will continue to be delivered though our respective technology transfer offices, with whom the principal applicants have strong links. The principal applicants have 14 patents between them and are experienced in spotting commercial opportunities from basic research and delivering potential licensing agreements as and when appropriate opportunities arise. The principal applicants have wide collaborations internationally and have been involved in clinical trials of novel antimicrobials and worked with companies ranging from SMEs to major pharmaceutical companies. The staff working on the project will develop team working skills which they can apply in all employment sectors, as well as writing skills, project management and investigation planning. Importantly, the scope for multidisciplinary training in this proposal should not be underestimated. The researchers employed to carry out the planned activities will have unique opportunities for hands-on experience across disciplines involved. Thus, our proposal will impact on the creation of human resources that could subsequently be employed in challenging interdisciplinary projects in industry, academia and government. We will communicate our findings to users and beneficiaries through publication in international peer-reviewed scientific journals, international conferences, university web pages and the press.