Plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β domain then forms a β-barrel pore in the outer membrane of the bacterium which allows the passenger domain to enter the external milieu. In some cases the passenger domain is cleaved from the β-barrel at the extracellular surface to release a soluble toxin. This is thought to be a self-contained process that does not require chaperones or ATP for folding and export of the passenger domain. Pet produces cytotoxic effects through cleavage of its target, the actin-binding protein α- fodrin. Pet is secreted into the extracellular environment, but its target lies within the cytosol. To reach its target, Pet moves from the cell surface to the ER where it triggers ER-associated degradation (ERAD) to enter the cytosol. ERAD is a normal cellular process in which improperly folded proteins are exported from the ER to the cytosol for degradation. Other toxins that utilize this pathway are AB toxins such as cholera toxin (CT) and ricin. The A subunits of these toxins are thermally unstable, and this facilitates their ERAD-dependent translocation into the cytosol. Pet, however, is not an AB toxin. We predict that thermal unfolding is not the mechanism Pet employs to exploit ERAD. It was necessary to purify the toxin first in order to study the structural properties and ER export of Pet. Surprisingly, purified Pet eluted as two close peaks by size exclusion chromatography. Both peaks were Pet as demonstrated through immunoblotting. The folding efficiency of autotransporters has not been extensively elucidated, and based on our purification results, we hypothesized that there is inefficiency in the folding of autotransporters, specifically Pet. A toxicity assay showed that Pet peak one did not display cytopathic activity while Pet peak two did. CD and fluorescence spectroscopy measurements also detected structural differences between the two variants of Pet and demonstrated that Pet peak one was an unfolded variant of Pet peak two. Native gel electrophoresis and biophysical measurements indicated that Pet peak one did not exist as a dimer or aggregate. Our results indicate there are two forms of Pet, and thus the folding process of autotransporters appears to be inherently inefficient. Active Pet (peak two) was used for further biophysical measurements and biochemical assays. Circular dichroism and fluorescence spectroscopy showed that the secondary and tertiary structures of Pet are maintained at physiological temperature, 37°C. Thermal unfolding of Pet occurred at temperatures above 50°C. Fluorescence quenching of Pet was also performed and demonstrated that, at 37°C, there are solvent-exposed aromatic amino acids. The slight structural alterations to Pet at physiological temperature as well as the exposed hydrophobic residues could trigger ERAD. In addition, a modeled structure of Pet revealed a hydrophobic loop which is surface-exposed and a likely target for toxin-ERAD interactions. The data suggests that translocation of Pet mediated by ERAD can occur by a mechanism different from certain AB toxins. An open, hydrophobic conformation likely triggers ERAD, but may also contribute to poor folding.

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As serino-proteases autotransportadoras de Enterobacteriaceae (SPATE) constituem uma família de proteases secretadas pelo sistema de secreção do tipo V, cujos genes foram estudados em Escherichia coli intestinal e extra-intestinal. Sat é uma SPATE citotóxica de 107 kDa, cujo gene foi identificado pela primeira vez em UPEC isolada da urina de um paciente com pielonefrite. A maioria dos estudos envolvendo Sat foram realizados em células renais e da bexiga, embora seu gene seja encontrado em DAEC, EAEC e, mais recentemente, em amostras bacterianas isoladas de septicemia neonatal e meningite. Os objetivos deste trabalho foram: i) purificar Sat; ii) determinar a ação da Sat purificada em diferentes tipos celulares e iii) caracterizar o papel de Sat na infecção in vitro por EAEC. Desta foram, a presença de Sat nos sobrenadantes do cultivo das cepas EAEC CV323 e DEC/Sat, isoladas de diarreia, foi confirmada por LC-MS/MS. Sat foi purificada da cultura de DEC/Sat+ e utilizada para a obtenção de anticorpos anti-Sat em coelho. O efeito citotóxico de Sat purificada foi investigado em células derivadas do endotélio (HUVEC) e do sistema urinário (Y1, LLC-PK1 e HEK-293) e gastrointestinal (Caco-2). Os parâmetros citotóxicos analisados foram: o descolamento celular e alterações na morfologia, permeabilidade e metabolismo mitocondrial das células. Para investigar o papel de Sat na infecção por EAEC, células Y-1 foram infectadas com EAEC CV323 e DEC/Sat+ na presença ou ausência de PMSF (inibidor de serino-protease) e anticorpos anti-Sat. Os parâmetros de citotoxicidade analisados nas culturas infectadas foram descolamento celular e alteração na morfologia. Os resultados demonstraram que: i) Sat é secretada por EAEC CV323 e DEC/Sat+ e, em ambas as cepas, há duas mutações em resíduos de aminoácidos que não interferiram na atividade enzimática; ii) as células do endotélio são mais sensíveis à Sat do que as células derivadas do trato urinário, sendo a linhagem gastrointestinal a mais resistente; iii) Sat secretada por EAEC CV323 durante a infecção induziu intenso dano celular, o qual, em presença de anticorpos anti-Sat e PMSF foi reduzido em cerca de 80 a 90%, respectivamente. Este é o primeiro trabalho que demonstra a expressão de Sat pela EAEC e a ação da toxina em células endoteliais sugerindo que o papel de Sat possa ser mais amplo na patogenia do que o proposto até o momento. / The serine protease autotransporters of Enterobacteriaceae (SPATEs) constitute a family of proteases secreted by the type V secretion system whose genes have been studied in intestinal and extra intestinal Escherichia coli. Sat is a 107 kDa cytotoxic SPATE and its gene was first identified in UPEC isolated from the urine of a patient with pyelonephritis. Most studies involving Sat were performed in renal and bladder cells, although the gene encoding Sat is encountered in other strains of E. coli such as DAEC, EAEC and more recently, in bacterial samples isolated from neonatal septicemia and meningitis. The objectives of this work were: i) purify Sat; ii) to determine the action of Sat in different types of cells and iii) to characterize in vitro the role of Sat in EAEC infection. Accordingly, the presence of Sat in the culture supernatant of EAEC CV323 and DEC/Sat+ derived from diarrhea was confirmed by LCMS/MS. Sat was purified from the culture of DEC/Sat+ and utilized to produce rabbit antibodies anti-Sat. The cytotoxic effect of Sat was investigated in cells derived from the endothelium (HUVEC) and the urinary (Y1, LLC-PK1, HEK-293) and gastrointestinal (Caco-2) systems. The cytotoxic parameters analyzed were cellular detachment and alterations in the morphology, permeability and mitochondrial metabolism of the cells. To investigate the role of Sat in EAEC infection, Y-1 cells were incubated with EAEC CV323 and DEC/Sat+ in the presence or absence of PMSF (a serine protease inhibitor) and rabbit antibodies anti-Sat. The parameters analyzed were cellular detachment and alteration in the morphology of the cells. The results demonstrated that: i) Sat is secreted by EAEC CV323 and DEC/Sat + and in both strains there are two mutations in amino acid residues that did not interfere with enzymatic activity; ii) endothelium cells are more sensitive to the Sat effect than the cells derived from urinary tract system, being the gastrointestinal cell lineage the most resistant one; iii) Sat secreted by EAEC CV323 during infection induced intense cellular damage which in the presence of anti-Sat antibodies and PMSF was reduced in about 80 to 90%, respectively. This is the first work demonstrating the expression of Sat by EAEC and the action of the toxin on endothelial cells suggesting that the role of Sat may be broader in pathogenesis than has been proposed so far.

A principal ecological role proposed for the hyporheic zone is as a refugium that promotes benthic invertebrate survival during adverse conditions in the surface stream. Whilst a growing body of work has examined use of this hyporheic refugium during hydrological extremes (spates, streambed drying), little research has considered variation in refugium use over prolonged periods including contrasting conditions of surface flow. In this thesis, benthic invertebrate use of the hyporheic refugium is considered at monthly intervals over a five-month period of variable surface flow, at nine sites in two groundwater-dominated streams, the River Lathkill (Derbyshire) and the River Glen (Lincolnshire). Conditions identified as potential triggers of refugium use included a flow recession and a high-magnitude spate on the Lathkill, and small spates and a decline in flow preceding localised streambed drying on the Glen. During flow recession, reductions in submerged habitat availability and concurrent increases in benthic population densities were dependent on channel morphology. An unusual paired benthic-hyporheic sampling strategy allowed the type of refugium use (active migration, passive inhabitation) to be inferred from changes in hyporheic abundance and the hyporheic proportion of the total population. Using this approach, evidence of active migrations into the hyporheic zone use was restricted to two instances: firstly, Gammarus pulex (Amphipoda: Crustacea) migrated in response to habitat contraction and increased benthic population densities; secondly, migrations of Simuliidae (Diptera) were associated with low-magnitude spates. Refugium use was site-specific, with refugial potential being highest at sites with downwelling water and coarse sediments. A conceptual model describing this spatial variability in the refugial capacity of the hyporheic zone is developed for low flow conditions. In some cases, hyporheic refugium use was apparently prevented by disturbance-related factors (rapid onset, high magnitude) regardless of the refugial potential of the sediments. The extension of the hyporheic zone's refugial role to include low flows highlights the need to explicitly protect the integrity of hydrologic exchange in river rehabilitation schemes. However, the limited capacity of the hyporheic refugium emphasizes the additional importance of maintaining habitat heterogeneity including multiple instream refugia.