Analysis of the binding properties and functions of scabies mite serine proteases

Reynolds, Simone Louise (2012). Analysis of the binding properties and functions of scabies mite serine proteases PhD Thesis, School of Veterinary Science, The University of Queensland.

       
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Author Reynolds, Simone Louise
Thesis Title Analysis of the binding properties and functions of scabies mite serine proteases
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Katja Fischer
Malcolm Jones
Angela Mika
Dave Kemp
Total pages 151
Total colour pages 11
Total black and white pages 140
Language eng
Subjects 110707 Innate Immunity
110803 Medical Parasitology
Formatted abstract
Scabies is a parasitic skin infestation caused by the mite Sarcoptes scabiei. Common worldwide, it remains a major public health problem in socially disadvantaged populations, including Australian indigenous communities. Scabies is a primary risk factor for superficial infections with pyogenic bacteria (pyoderma) and there is increasing recognition of the significant health burden caused by scabies and pyoderma. With poor treatment success and growing epidemiological evidence that scabies is linked to pathogenic bacterial disease, the underlying molecular mechanisms present a novel target for intervention.

A family of proteolytically inactive serine protease paralogues (SMIPP-Ss) has been implicated in host defence evasion. Studies of the SMIPP-S family reveal that at least five SMIPP-Ss function as inhibitors of the host complement system, presumably to prevent complement-mediated gut damage. To assess this role in host defence evasion two SMIPP-Ss (D1 and I1) were extensively studied and are the focus of this thesis. The study revealed that SMIPP-Ss D1 and I1 bind to complement factors C1q, mannose binding lectin and properdin. The biological relevance of these findings was reinforced by the localisation of complement C1q in the mite gut.

To understand the nature of the binding to complement factors, previously produced crystal structures of SMIPP-Ss D1 and I1 were used to identify potential binding sites. The structures had revealed that occlusion of the S1 subsite by a conserved tyrosine blocked substrate access. It was concluded that the SMIPP-Ss had “lost” the ability to bind substrates in a classical fashion and had evolved alternative sites of interaction. The overlaying of thirty SMIPP-S sequences, on the two structures, revealed areas of high conservation, which represented potential functional exosites.

To determine functional sites of interaction, the information from the structural studies was utilised to design a series of mutants. Employing site directed mutagenesis N-linked glycans were added into regions of conservation and mutant proteins were tested for loss of inhibitory function in complement assays. Introducing glycosylation in close proximity of K108 of SMIPP-S I1 and K103 of SMIPP-S D1 resulted in reduced complement inhibition. Finer mapping, by introducing specific residue changes, confirmed that this region is involved in binding to mannose binding lectin in both SMIPP-Ss. To rule out that loss of function in mutants resulted from a loss in conformation, proteins were subjected to Circular Dichroism Spectroscopy. This confirmed that structural integrity had been maintained.

In tropical climates the association between scabies and pyoderma caused by S. pyogenes (GAS) and S. aureus has been well established. Microbes associated with scabies are vulnerable to complement attack and have evolved their own defence mechanisms. To investigate if the anti-complement activity of SMIPP-S is beneficial to bacteria functional studies were conducted. Using whole blood bactericidal assays containing human phagocytes and complement it was found that the presence of either SMIPP-S D1 or I1 increased Group A Streptococcal survival by up to 15-fold in vitro. This study suggests that a molecular mechanism contributes to the positive association between scabies and Group A Streptococcal skin infection.

This thesis represents an extensive study of two SMIPP-Ss from the SMIPP-S multi-gene family. SMIPP-Ss D1 and I1 are potent inhibitors of the complement system with the ability to bind to the complement components C1q, mannose binding lectin and properdin. Site directed mutagenesis studies have revealed the binding interaction site on both SMIPP-Ss for mannose binding lectin. Additional functional studies investigating the role of SMIPP-Ss in bactericidal assays have shown that in the presence of either SMIPP-S D1 or I1 streptococcal survival rates are significantly improved. This thesis proposes that the co-existence of scabies mites and bacteria results in complement inhibition which promotes survival of scabies mites and bacterial pathogens in skin burrows. The emerging resistance of mites and bacteria to current therapeutics emphasises the need to understand the underlying molecular mechanisms and to identify novel targets for intervention.
Keyword Scabies
Complement
Immune evasion
Serine protease
Indigenous health
Staphylococcal
Streptococcal

 
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Created: Fri, 14 Dec 2012, 09:38:22 EST by Simone Reynolds on behalf of Scholarly Communication and Digitisation Service