Investigating the conditions that trigger filamentation in uropathogenic Escherichia coli

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Urinary tract infections (UTIs) are one of the most common and costly infections worldwide, primarily caused by Uropathogenic Escherichia coli (UPEC). Over recent years, there has been an increase in antibiotic resistant UTIs, which are diminishing the use of existing antibiotic therapies. This requires close surveillance as it has the possibility to greatly exacerbate the impact of UTIs. Therefore, there is a high need to develop new treatments that can replace current ineffective antibiotic therapies. In order to identify new treatment options, the different stages of UPEC infection need to be understood. UPEC undergo a multi-stage infection cycle, which is initiated by UPEC binding to and being internalised into host bladder epithelial cells that line the bladder wall. The bacteria continue to grow inside the bladder cells where they develop into intracellular bacterial communities, which are internal biofilm-like colonies. Once the bacterial burden has overwhelmed the host, the bladder cell ruptures and releases the bacteria. A substantial proportion of the released bacteria consist of extensively elongated bacteria, many times longer than typical rod-shaped E. coli. This morphology change is known as filamentation, where the bacterial cell has continued to grow without dividing. This occurrence has been thought to offer survival advantages to the bacteria to allow it to cause such a successful and persistent infection. Studies have demonstrated that one trigger that induces bacterial filamentation is concentrated urine, although the underlying mechanisms are unknown. This thesis aimed to further define and investigate the conditions and factors that trigger bacterial filamentation in a UTI. Initially, a method was required to accurately quantify the degree of filamentation by reproducing and improving on current in vitro bladder cell infection models. Through the development of an appropriate fluorescent UPEC strain to visualise the bacteria during infection of human bladder cells, a reproducible method, based on the combined use of microscopy and flow cytometry, was established to determine the degree of filamentation under different conditions. By directly visualising the events before and during bacterial filamentation, it can develop an understanding of how and why filaments arise. Therefore, a novel microfluidic infection model was established to observe the infection of bladder cells by UPEC in real time. This model revealed the bacteria do not initially inactivate or kill their host cells, but appear to use the bladder cells as protection to grow and develop. With this new model it was also demonstrated that cultured human bladder cells became immobilised, permeabilised and likely killed upon exposure to a flow of sterile human urine. This was unexpected but revealed a likely mechanism by which the bacteria could rapidly respond to form filaments, from inside the host bladder cells. Nevertheless, it was expected that the exposure to urine was a key factor that triggers the filamentation response of UPEC after bladder cell rupture both in vitro and in vivo. For the first time, UPEC filaments were directly observed to grow out from within permeabilised bladder cells after exposure to urine. To begin to understand what initiates bacterial filamentation in UTI potential factors were investigated. The well-characterised SOS response was analysed for its importance to UPEC filamentation. This response is known to cause filamentation after bacterial DNA damage and there has been some conflicting reports over the role of SOS filamentation in a UTI. The work in this thesis indicated that it is unlikely that SOS induction is the primary cause of filamentation in a UTI. Investigations were conducted into host factors, such as the composition of urine, as a condition that induces bacterial filamentation. The results showed that filamentation occurs in response to a certain urine constituent(s), that is pH dependent and of small molecular weight. By developing and utilising in vitro infection models of UTIs, this thesis has initiated a new line of research into the effects of different factors, both bacterial (SOS) and host related (urine), on filamentation. Overall, this thesis provided evidence of the existence of other non-SOS pathways that are sensitive to urine composition that cause UPEC to filament during infection. This knowledge could be used in the future to help develop treatments that focus on preventing filamentation and the infection cycle, in the hope of attenuating this common infection.
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