View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Queensland University of Technology ePrints Archive The role of endogenous scabies mite complement inhibitors in the development of Streptococcus pyogenes skin infections Lindsay Darryl Christian Bachelor of Applied Science (Biotechnology) Submitted in partial fulfilment of the requirements for the degree of Master of Applied Science (Research) School of Biomedical Sciences Institute of Health and Biomedical Innovation (IHBI) Faculty of Health Queensland University of Technology (QUT) & QIMR Berghofer Medical Research Institute August 2015 Keywords Scabies; Streptococcus pyogenes; secondary bacterial infection; complement; opsonisation; phagocytosis ii Statement of Originality The work presented in this thesis was carried out at the QIMR Berghofer Medical Research Institute under the supervision of Dr Katja Fischer and Dr Pearl Swe. Professor Louise Hafner and Professor Peter Timms were the supervisors at the Queensland University of Technology. This material has not previously been submitted for an award at any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made. QUT Verified Signature Signed: Date: ____________________ iii Acknowledgements I would like to thank Professor Louise Hafner, Professor Peter Timms (School of Biomedical Sciences, Queensland University of Technology), Dr Katja Fischer and Dr Pearl Swe (Scabies group, QIMR Berghofer Medical Research Institute) for their supervision during my Masters studies. Thank you Louise for insisting on regular meetings to keep me on track and for always being available and patient with me when I needed guidance. Likewise, thank you Pearl and Katja for your patience and the endless hours spent mentoring and training me in many of these finicky lab techniques. I’d like to thank Dr Simone Reynolds (Scabies group, QIMR Berghofer Medical Research Institute), Professor Sri Sriprakash and Dr Celia McNeilly (Bacterial Pathogenesis laboratory, QIMR Berghofer Medical Research Institute) for providing their expert advice and feedback on experimental techniques and during meeting presentations. I acknowledge the time and assistance of Dr Tam Hong Nguyen (Flow Cytometry and Imaging Facility, QIMR Berghofer Medical Research Institute), for help with the fluorescent microscopy; Dr Grace Chojnowski (Flow Cytometry and Imaging Facility, QIMR Berghofer Medical Research Institute) for training me on Flow cytometry; and Fabian Riveria (Immunology and Infection laboratory, QIMR Berghofer Medical Research Institute) for assistance with data analysis on FlowJo. Thank you also to Associate Professor David Leavesley and Associate Professor Rob Harding (Queensland University of Technology) for reviewing my thesis draft and providing valuable feedback during my final seminar. I am grateful for the experiences I’ve had during my time on this project. Highlights included; attending and presenting at the 2014 Australian Society of Parasitology conference in Canberra and assisting with a Lowitja Institute ‘Knowledge exchange’ workshop at Cloncurry High School, where along with the Scabies group, I contributed to introducing high school students to how interesting and important health research can be. I am also appreciative for being a recipient of the Heart Foundation Australia Indigenous postgraduate scholarship that has provided me with financial support during my Masters. Thank you again to my supervisors for encouraging me to apply and participate in these opportunities. Lastly, I’d like to thank Claire; for her support throughout my study journey. iv Abstract Scabies and associated bacterial skin infections are highly prevalent in the tropics and a major disease burden in Australian Aboriginal communities. It is believed that damage to the upper layers of the human skin as a result of the burrowing action of scabies mites provides an entry point for bacterial pathogens. One of the most frequently associated bacterial pathogen with scabies infections is Streptococcus pyogenes or Group A Streptococcus (GAS). This is of serious concern because of the potentially fatal secondary diseases associated with GAS infections such as acute rheumatic fever (ARF), rheumatic heart disease (RHD) and post- streptococcal glomerulonephritis (PSGN). In Indigenous Australian communities the prevalence of ARF/RHD is amongst the highest rates reported globally. Traditionally, ARF/RHD was attributed to GAS infections of the throat; however, GAS throat infections are relatively rare in many communities that have high rates of ARF/RHD, while GAS skin infections are common in these communities. Previous studies have shown that scabies mites secrete proteins that inhibit the host’s immune defence processes, thereby potentially promoting the survival of pathogenic bacteria in these hosts. These initial studies indicated that scabies mite infections may play important roles in the establishment, proliferation and transmission of a range of microbial pathogens. This project aimed to address the mechanisms by which the scabies mite complement inhibitors, inactivated protease paralogue - serine I1 (SMIPP-S I1) and serpin B4 (SMS B4) - promote the recovery of GAS. A variety of clinical GAS strains were examined in this study, including S. pyogenes 88-30; isolated from a scabies skin lesion of an Aboriginal Australian in the Northern Territory. The methodology included global bactericidal assays, deposition assays and phagocytosis assays to demonstrate the effects of the mite complement inhibitors on complement killing of bacteria, complement deposition on the bacteria surface and phagocytosis of bacteria, respectively. It was found that both SMIPP-S I1 and SMS B4 proteins promoted the growth of S. pyogenes in a concentration- dependent manner, and that these effects were not GAS strain-specific. The scabies mite complement inhibitors were found to interfere with the activation and progression of the Classical and Alternative pathways of the complement system. Most notably, both mite complement inhibitors reduced the opsonisation of GAS strains by decreasing the deposition of C3b on the bacterial cell surface. These findings indicate that scabies mite complement inhibitors may play key roles in establishing pyoderma, or purulent skin diseases caused by infections with pathogens such as S. pyogenes. This novel insight at a molecular level into the pathogenesis of scabies and secondary bacterial infections may provide a new explanation why v patients infected with scabies are predisposed to secondary bacterial infections, potentially leading to severe and fatal secondary diseases such as PSGN, ARF and RHD. Tabl e of Content s vi Tabl e of Cont ent s Tabl e of Cont ent s Table of Contents Keywords ................................................................................................................................... ii Statement of Originality.............................................................................................................iii Acknowledgements ................................................................................................................... iv Abstract ...................................................................................................................................... v Table of Contents ......................................................................................................................vii List of Abbreviations ................................................................................................................. x List of Tables, Figures, and Equations..................................................................................... xiii Chapter 1: Introduction .............................................................................................................. 1 1.1 The scabies mite .......................................................................................................... 2 1.2 Clinical presentations of scabies ................................................................................. 3 1.3 Epidemiology of scabies ............................................................................................. 4 1.4 Diagnosis, treatment and control of scabies................................................................ 4 1.5 Secondary bacterial infections .................................................................................... 5 1.6 Rheumatic heart disease (RHD) .................................................................................. 6 1.7 GAS pyoderma and RHD............................................................................................ 7 1.8 Molecular research into the biology of the scabies mite ............................................. 7 1.9 The host innate immune response to scabies .............................................................. 8 1.10 Pathogen serpins and inactive proteases ................................................................... 10 1.11 Scabies mite complement inhibitors ......................................................................... 10 1.12 Skin damage, complement inhibition and the cutaneous microbiome...................... 12 1.13 Significance ............................................................................................................... 13 1.14 Aims and Hypothesis ...............................................................................................