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Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with severe nosocomial infections in immunocompromised hosts and patients with cystic fibrosis (CF). During infection the bacteria secrete proteins that are essential to the infection process. Several of these virulence-associated proteins have been identified using genetic methods. The aim of this research, using a proteomic approach, was to identify novel extracellular proteins that are secreted by P. aeruginosa during infection of a CF patient. Extracellular proteins from P. aeruginosa strain PAO1 grown in vitro were separated by two-dimensional gel electrophoresis (2-DE). The humoral response of chronically infected CF patients to the separated proteins was elucidated using western blotting. Growth phase, cell population and iron limitation were identified as important regulators of the extracellular proteome. The number of extracellular proteins significantly increased upon entry into stationary phase, as did the number of proteins detected by CF patient sera. The detection of several known quorum-controlled proteins by patient sera indicated the importance of this regulatory mechanism during infection. In iron-limiting medium, the proportion of proteins detected by CF patient sera significantly increased compared to extracellular proteins from cells grown in iron-replete conditions. Proteomic analysis of a PAO1 pvdS mutant strain showed that PvdS (an iron-regulated alternative sigma factor) directs production of many extracellular proteins made during infection of a CF patient. Examination of extracellular proteins from a second strain, PA4, indicated it had a shared set of extracellular proteins. The identities of selected proteins were determined and these included well-characterised extracellular virulence factors such as elastase (LasB). Also identified were proteins with a potential virulence role such as azurin (a copper containing redox protein), PA2939 (a likely aminopeptidase) and proteins with unknown functions. This study provides the first evidence for the production of these proteins during infection. In summary, the proteomics methodology developed here facilitated the rapid identification and enumeration of proteins secreted by P. aeruginosa during infection.

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Geobacillus thermoglucosidasius (GT) is a thermophilic, ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. One strategy to improve fermentation yields would be to engineer GT strains to secrete hydrolases to increase the amount of available sugars from various feedstocks. Therefore, optimised protein secretion would be vital to improve feedstock utilisation. Secretion in the related mesophile Bacillus subtilis (BS) has been well studied, and several strategies have been developed to improve secretion of heterologous proteins in BS, one such strategy being the manipulation or changing of the signal peptide. One aim is to identify any differences in the secretion machinery and signal sequences between GT and BS. Another aim is to analyse any effects of overproduction of hydrolases and to identify any bottlenecks in protein secretion in GT. Using bio-informatics tools we find that although GT is a thermophile, the signal peptides in this organism do not differ significantly from those in BS. From a shotgun mass spectrometry approach it was also observed that unlike BS, GT undergoes significant cell lysis during growth releasing cytoplasmic proteins into the extracellular milieu, which could have implications on the levels of secreted hydrolases. A model enzyme was selected and over-produced at high levels in order to stress the secretion system in GT so as to identify any bottlenecks in secretion. The results thus far indicate that the rate limiting step in secretion could be post-translocation where the enzyme is degraded by proteases in the cell wall and extracellular milieu. The addition of protease inhibitor to growth media, increases the activity and abundance of the enzyme, suggesting that proteolysis may be a major factor when over-producing secreted enzymes at high levels.

Enterotoxigenic Escherichia coli (ETEC) encompass a group of diverse bacterial pathogens that collectively cause hundreds of millions of diarrheal cases annually, mostly in developing countries. As part of its infection strategy, ETEC invades and colonizes small intestinal epithelial cells where it secretes heat-labile and/or heat-stable enterotoxins, inducing diarrhoea. The ability of ETEC to invade human epithelial cells is a hallmark of its pathogenicity and is controlled by a set of plasmid and chromosome encoded virulence factors. They include EtpA, a 170 kDa plasmid encoded autotransporter. During infection, EtpA functions as an adhesin linking flagellin at the tip of ETEC flagella to the host cell surface and allowing ETEC to deposit its toxins. Antibodies targeting either EtpA or the conserved regions of flagellin impair delivery of the heat-labile toxin in vitro, and prevent intestinal colonization of mice following gastrointestinal challenge with ETEC. EtpA is thus critical to the pathogenicity of ETEC. In this study, a truncated version of EtpA (35 kDa) termed N-terminal EtpA69-445 or N-EtpA69-445 was cloned and produced as an N-terminal GST-tagged cytoplasmic fusion protein in E. coli BL21 cells. The protein was purified by affinity chromatography on glutathione agarose beads. However, the yield of the pure protein was poor due to its limited solubility. As an alternative, a 57 kDa truncated version of EtpA (N-EtpA69-607) was produced as a secreted C-terminal His6-tagged fusion protein in E. coli TOP10 cells. The protein was purified to homogeneity by metal affinity chromatography (MAC) using Ni-NTA and ion exchange chromatography (IEC) on a Mono S 10/100 GL column. Biophysical characterization of N-EtpA69-607 using circular dichroism (CD) spectroscopy revealed the typical spectrum of a β-helical protein. The in silico modelled structure of the protein confirmed N-EtpA69-607 to be a β-helical protein. CD spectra recorded at increasing temperatures indicated N-EtpA69-607 to be thermally highly stable retaining its fold up to 95°C. Dynamic light scattering (DLS) experiments showed that N-EtpA69-607 is polydisperse in solution forming higher oligomers. Lead crystallization conditions of N- EtpA69-607 were determined but the crystals were too small for X-ray data collection. This study thus represents a significant step towards the characterization of the three dimensional structure of EtpA and understanding its structure-function relationship. / Dissertation (MSc)--University of Pretoria, 2016. / Biochemistry / MSc / Unrestricted