Investigation of the molecular basis of Salmonella Typhimurium Intestinal persistence

The bacterium Salmonella accounts for about 125 million incidents of disease worldwide each year, and nearly a million deaths. The morbidity and mortality caused by this pathogen has a significant impact on the economies of both resource rich and resource poor countries. Most cases of non-typhoidal Salmonella result from fecal contamination of food and food products. A common and therefore critical step for this is the entry of the bacterium into the food chain from livestock and poultry, the common zoonotic reservoir for this pathogen.
While a large body of literature describes the mechanisms by which Salmonella establishes infection in the intestine, comparatively little is known about how Salmonella persists. Decreasing the amount of Salmonella in animals at slaughter is important to controlling Salmonella food-borne disease because it is the primary risk factor for introduction of this pathogen into the food chain destined for human consumption. Salmonella uses distinct mechanisms for initial colonisation and persistent colonisation and the latter represent novel targets for approaches to decrease the amount of Salmonella in livestock when they arrive at slaughter. However, in order to develop new approaches it is essential to first identify how Salmonella remains associated with the intestine in the face of competition from good bacteria resident in the intestine and the response by the host animal aimed at clearance of bacterial pathogens. This programme of work will identify all the genes encoded by Salmonella that are involved in the ability of this pathogen to remain in the intestine of livestock for prolonged periods. The relative importance of newly discovered persistence factors in this study and the previously described persistence factors ShdA and MisL will be determined in order to inform the optimal strategy for the design of intervention to decrease Salmonella colonisation. ShdA and MisL are found on the surface of Salmonella where they are known to interact with host proteins but exactly how this contributes to prolonged colonisation of the intestine is not known. We will determine how ShdA and MisL contribute to intestinal persistence, extending on a large body of published work. We will use a combination of an in vivo model of intestinal persistence developed by the applicant, studies on animal cells grown in the lab to determine the impact of ShdA and MisL on interaction of Salmonella with the host, and investigate how these bacterial proteins interact with proteins of the host. The outcome of this study will be a comprehensive overview of the intestinal persistence factors encoded by Salmonella and an understanding of the molecular basis of intestinal persistence mediated by ShdA and MisL. This data analyses will complement efforts by the livestock industry to devise strategies to decrease the incidence of Salmonella carriage that include farm-yard practice and the potential implementation of a vaccine against Salmonella.

The overall aim of this programme of work is to investigate mechanisms of intestinal persistence of Salmonella Typhimurium (Stm). We will address the central hypothesis that a subset of genes encode intestinal persistence factors including ShdA and MisL that are involved in mechanisms contributing to intestinal persistence in the murine host. The significance of this work is that an understanding of mechanisms by which Salmonella persists in the intestine of zoonotic animal reservoirs will lead to the rational design of intervention strategies aimed at preventing the entry of pathogens into the food chain. This programme builds on a considerable body of the PI's published and preliminary data that revealed i) ShdA and MisL contribute to intestinal persistence in mice, calves and pigs, ii) ShdA and MisL bind Fn in vitro, iii) Salmonella infection results in increased expression of Fn in areas of the intestine colonised by Stm, iv) coating of Stm with Fn enhances invasion of epithelial cells in culture, v) ShdA is a rod structure composed of beta-helices and interacts specifically with a cationic cradle in the 13FnIII repeat, vi) mAbs recognising a specific epitope in the A3 repeat of ShdA inhibits fibronectin (Fn) binding, and vi) ShdA interacts with a complex of host proteins involved in pathways of pathogen invasion. Specifically we will determine 1) the essential genes for persistence in the mouse intestine, 2) the impact of ShdA binding to Fn on the distribution and persistence of Salmonella in the murine cecum, and 3) the impact of ShdA / MisL on the interaction with host cells and host protein complexes. The outcome of this study will be a comprehensive overview of the intestinal persistence factors encoded by Salmonella and an understanding of the molecular basis of intestinal persistence mediated by ShdA and MisL.

The beneficiaries of this research include research scientists interested in mechanisms of host-pathogen interactions, the livestock and food processing industries interested in novel methods to decrease the presence of zoonotic pathogens in livestock entering the food chain and the National Health Service and the general public that will benefit from efforts to decrease foodborne disease.
1. Research scientists. There is considerable interest in the scientific research community in molecular mechanisms of host-pathogen interactions. While a considerable amount of literature already exists on how bacterial pathogens engage with the host to establish infection and cause disease, comparatively little is known about how they remain associated with the host in order to maximise transmission. In particular the combination of a relevant infection model and cell biology to elucidate the molecular mechanisms of intestinal persistence factors in this programme of work will significantly contribute to this gap in knowledge. Although the present study will focus on Salmonella, many other pathogens of economic importance such as Campylobacter spp. face similar challenges to persistently colonise host animals and indeed encode adhesins with similar activity to those to be addressed in this study. It is therefore anticipated that an understanding of how Salmonella maintains persistent colonisation in livestock will provide insight more widely applicable to researchers interested in related zoonotic pathogens.
2. Livestock and food processing industries. The food industry and in particular the pig rearing industry is focussing on the need to decrease the incidence of Salmonella in the food chain. There is a need for livestock farms and abattoirs to decrease the incidence and levels of Salmonella in animals arriving for slaughter in order to decrease the likelihood that the pathogen will enter the food chain. The pig rearing industry is committed to decreasing the incidence of Salmonella in pig herds as part of an obligation set out by EU legislation within the Zoonoses National Control Programme (ZNCP), which from June 2014 requires abattoirs to identify farms where pigs are frequently contaminated with Salmonella. The output from this project will provide the knowledge basis for informing the on-farm practices to achieve this and the development of effective intervention strategies. For example, there is current interest in using a vaccine to reduce the carriage of Salmonella in pig herds and a current study led by Dr Rob Davies at AHVLA is testing the immune response to a candidate vaccine. Our study will identify important determinants of intestinal persistence that if targeted by the vaccine may significantly decrease the amount of salmonella present in livestock at slaughter.
3. National Health Service and the general public. The longer term aim of this programme of work will benefit the general public by improving the supply of safe food products with reduced levels of zoonotic pathogens. The cost of foodborne disease in the UK each year is estimated to be around £1.2 billion (www.food.gov.uk), highlighting the need to improve the safety of our food supply and the potential benefit to society of doing so.
STRATEGIC RELEVANCE: The proposal addresses gaps in the fundamental knowledge that impacts the ability of the UK food industry to deliver safe food with a reduced risk of contamination with food borne pathogens, in particular Salmonella. It is therefore directly relevant to the BBSRC strategic priorities 'Food, nutrition and Health'.