New prophylactic and therapeutic treatments to combat pathogenic Enterohaemorrhagic Escherichia coli

Bacterial diarrhoeal diseases have significant influence on global human health, and are a leading cause of preventable death in the developing world. Enterohaemorrhagic Escherichia coli (EHEC), pathogenic strains of E. coli that carry potent toxins, have been associated with a high number of large-scale outbreaks caused by contaminated food and water sources. This pathotype produces diarrhoea and haemorrhagic colitis in infected humans, and in some patients leads to the development of haemolytic uremic syndrome (HUS), which can result in mortality and chronic kidney disease. A major obstacle to the treatment of EHEC infections is the increased risk of HUS development that is associated with antibiotic treatment, and rehydration and renal support are often the only options available. New treatments designed to prevent or clear E. coli infections and reduce symptoms of illness would therefore have large public health and economic impacts. The three main aims of this thesis were: to explore mouse models for pre-clinical evaluation in vivo of small compounds that inhibit a major EHEC colonisation factor, to assess the production and role of two proteins considered promising candidates for a broad-spectrum vaccine against pathogenic E. coli, and to investigate a novel compound that has recently been identified as a potential inhibitor of EHEC toxin production. As EHEC cannot be safely tested in humans due to the risk of HUS development, appropriate small animal models are required for in vivo testing of new drugs. A number of different mouse models have been developed to replicate different features of EHEC pathogenesis, several of which we investigated with a focus on colonisation mediated by the Type III Secretion System (T3SS), a needle-like structure that translocates bacterial proteins into host cells, resulting in a tight, intimate attachment between pathogen and host, aiding colonisation of the gastrointestinal tract. As E. coli models were found not to depend significantly on the T3SS for colonisation, the Citrobacter rodentium model, a natural mouse pathogen closely related to E. coli, was deemed the most suitable mouse model currently available for in vivo testing of T3SS-targeting compounds. Two bacterial proteins, EaeH (an outer membrane adhesin) and YghJ (a putative secreted lipoprotein), highly conserved surface-associated proteins recently identified as III protective antigens against E. coli infection of mice, were explored in order to determine their suitability as candidates for a human vaccine against pathogenic E. coli. We focused on the expression and function of these proteins in the EHEC O157:H7 EDL933 strain and the adherent-invasive E. coli (AIEC) LF82 strain. Although expression of EaeH by other E. coli pathotypes has recently been shown to be upregulated upon contact with host intestinal cells, no evidence of this upregulation could be demonstrated in our strains. Additionally, while YghJ was produced by the AIEC strain, it was not secreted by bacteria under conditions that other YghJ-expressing E. coli pathotypes do, despite the AIEC strain carrying all the genes required to encode the secretion system it is associated with. While our findings indicate that a vaccine that raises antibodies against EaeH and YghJ may have limited effect on the EHEC and AIEC strains we used, recent studies into these proteins in different E. coli pathogens have suggested they are still excellent candidates for a broadly effective vaccine against E. coli. Finally, we characterised a small lead compound, identified by high-throughput screening as a possible inhibitor of Shiga toxin expression. Shiga toxin production causes both the symptoms of illness and development of HUS, and thus reduction of toxin production, release, or binding to host receptors could therefore be an effective way to treat infections and decrease the risk of HUS. Inhibition of Shiga toxin production by this compound was confirmed, and was shown to be caused by an inhibitory effect on activation of the bacterial SOS response rather than on the Shiga toxin genes themselves. The bacterial target of this compound was identified as RecA, a major regulator of the SOS response, and we hypothesise that the compound binds covalently to its target, preventing oligomerisation of RecA into an activated filament. Altogether, the results presented here provide an improved understanding of these different approaches to combating EHEC infection, which will aid the development of safe and effective vaccines and anti-virulence treatments against EHEC.

Dynamically managing sensed context data

This thesis reports on an investigation of the feasibility and usefulness of incorporating dynamic management facilities for managing sensed context data in a distributed contextaware mobile application. The investigation focuses on reducing the work required to integrate new sensed context streams in an existing context aware architecture. Current architectures require integration work for new streams and new contexts that are encountered. This means of operation is acceptable for current fixed architectures. However, as systems become more mobile the number of discoverable streams increases. Without the ability to discover and use these new streams the functionality of any given device will be limited to the streams that it knows how to decode. The integration of new streams requires that the sensed context data be understood by the current application. If the new source provides data of a type that an application currently requires then the new source should be connected to the application without any prior knowledge of the new source. If the type is similar and can be converted then this stream too should be appropriated by the application. Such applications are based on portable devices (phones, PDAs) for semi-autonomous services that use data from sensors connected to the devices, plus data exchanged with other such devices and remote servers. Such applications must handle input from a variety of sensors, refining the data locally and managing its communication from the device in volatile and unpredictable network conditions. The choice to focus on locally connected sensory input allows for the introduction of privacy and access controls. This local control can determine how the information is communicated to others. This investigation focuses on the evaluation of three approaches to sensor data management. The first system is characterised by its static management based on the pre-pended metadata. This was the reference system. Developed for a mobile system, the data was processed based on the attached metadata. The code that performed the processing was static. The second system was developed to move away from the static processing and introduce a greater freedom of handling for the data stream, this resulted in a heavy weight approach. The approach focused on pushing the processing of the data into a number of networked nodes rather than the monolithic design of the previous system. By creating a separate communication channel for the metadata it is possible to be more flexible with the amount and type of data transmitted. The final system pulled the benefits of the other systems together. By providing a small management class that would load a separate handler based on the incoming data, Dynamism was maximised whilst maintaining ease of code understanding. The three systems were then compared to highlight their ability to dynamically manage new sensed context. The evaluation took two approaches, the first is a quantitative analysis of the code to understand the complexity of the relative three systems. This was done by evaluating what changes to the system were involved for the new context. The second approach takes a qualitative view of the work required by the software engineer to reconfigure the systems to provide support for a new data stream. The evaluation highlights the various scenarios in which the three systems are most suited. There is always a trade-o↵ in the development of a system. The three approaches highlight this fact. The creation of a statically bound system can be quick to develop but may need to be completely re-written if the requirements move too far. Alternatively a highly dynamic system may be able to cope with new requirements but the developer time to create such a system may be greater than the creation of several simpler systems.