Doctor of Philosophy
School of Biological Sciences
Cork, Amanda Jane, Characterisation of the streptococcal surface proteins, SEN and GAPDH, and their interaction with host molecules, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2011. http://ro.uow.edu.au/theses/3249
Streptococcus pyogenes (Group A Streptococcus; GAS) is a Gram-positive pathogen that causes a wide range of infections and diseases in humans. These infections can range from the mild infections that are common such as pharyngitis and impetigo, to the more serious and rare infections such as necrotising fasciitis and rheumatic heart disease. Since the 1980s there has been a resurgence of GAS invasive infections in western countries, however the major disease burden lies in Indigenous populations and developing nations. Of great concern are the endemic outbreaks in Aboriginal populations of northern Australia. There has been a large body of research into the mechanism of invasive disease, however the pathogenesis of GAS is yet to be fully understood. GAS contains several virulence factors at the cell surface, and it is thought that interactions between these virulence determinants and host proteins may serve as a vehicle for the initiation and progression of streptococcal infection.
One host protein that is often implicated in streptococcal infection is the serine protease plasminogen. Plasminogen has the ability to degrade fibrin clots, components of the extracellular matrix, and contribute to cell migration. GAS has several plasminogen binding receptors at the cell surface, two of which are classically known for their role in the glycolytic pathway. These are streptococcal surface enolase (SEN) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and are the focus of this research.
In this study, SEN was expressed and purified using an E. coli expression system, then polyclonal antisera was raised to detect the protein in subsequent experiments. Functional studies on the purified recombinant protein found that it was enzymatically active as it was able to convert 2-phosphoglycerate to phosphoenolpyruvate at a rate of 0.123 μmol min-1 μg-1. The ability of SEN to bind Glu-plasminogen was also confirmed using ligand blot analysis and surface plasmon resonance, where binding to immobilised plasminogen of approximately 420 response units was observed. Using CDspectroscopy it was observed that SEN contained approximately 48% α-helices. Structural characterisation confirmed that like other bacterial enolases, SEN was found to exist as an octamer as determined by mass spectrometry, with a collision crosssection calculated to be 12,763 ± 580 Ǻ2. The octamer was comprised of a tetramer of dimers as depicted by homology modelling and the estimated cross-section of the molecule corresponded well with the measured value.
To further characterise the mechanism of plasminogen binding by SEN, several exposed lysine residues, as determined by molecular modelling, were chosen and substituted for another amino acid. Lysine residues were of interest as they are primarily implicated in the interaction with the kringle domains of plasminogen. Functionally, it was found that the enzymatic activity was retained for SENK252A+K255A, SENK261A, SENK304A, SENK312A, SENK362A and SENK435L, was reduced in SENK252A, SENK255A, SENK285A, SENK334A and SENΔ434- 435, and completely abolished in SENK344E. It was also observed that plasminogen binding ability was greatly diminished for SENK252A+K255A, SENK435L and SENΔ434-435, implying that these residues play a concerted role in the acquisition of plasminogen. Interestingly, the ability to bind human Glu-plasminogen apparently increased for SENK252A, SENK255A, SENK261A, SENK285A, SENK312A, SENK344E and SENK362A. By investigating the structural features of these mutants it was apparent that SENK344E was structurally unstable and this may afford the increase in plasminogen binding ability. Although overall structure in all other mutants was retained, small structural changes not detected by mass spectrometry that may cause relaxation of the octamer might explain the evident increase in plasminogen binding. Regardless, using structural analysis tools in tandem with functional studies highlights the advantage of structure/function analysis in revealing SEN-plasminogen interactions.
The ability of GAPDH to bind several mammalian proteins was also addressed in the work herein. Interaction with human proteins may play a role in the attachment of the bacterium to host tissues, aid in the avoidance of the human defence system, or serve as a mechanism to invade and colonise the host. The gapdh gene was successfully amplified from M1 GAS as a 1.15 kb fragment, and cloned into an E. coli expression system. GAPDH was purified and used to raise polyclonal antisera, which was used to detect the recombinant protein in proceeding experiments. GAPDH was found to bind actin and glu-plasminogen by ligand-blotting techniques. When using the more sensitive ELISA technique, GAPDH was found to also interact with egg-white lysozyme and human lysozyme. GAPDH was not observed to bind myosin or fibrinogen using either technique. The function of GAPDH-lysozyme binding was investigated using lysozyme activity assays, where it was concluded that the binding of lysozyme by GAPDH had no affect on its bacteriolytic ability. Further characterization both structurally and functionally of GAPDH with these mammalian proteins is required to fully understand the role of this multifunctional binding protein in the pathogenicity of GAS.
This work investigated the binding interaction of SEN and host plasminogen, using structural information to validate the observed binding function. The ability of GAPDH to interact with host proteins is also addressed, and the possible advantage of these interactions is discussed. By studying protein-protein interactions and fully understanding the mechanism of binding in terms of structure, therapeutics may subsequently be developed to circumvent infection and the diseases caused by GAS.