A molecular basis for autoimmune sequelae of streptococcal infections

The emergence of bacteria resistant to almost all known antibiotics is one of the most important challenges for science. If no new prevention and treatment options for bacterial infectious are developed we may soon slip back into the state of the 19th century when people died of what we very recently thought of as trivial infections. In order to develop vaccines or treatments that differ significantly from drugs like penicillin we need to understand better the ways in which bacteria cause infections. To start with, bacteria have to gain a foothold in the human body. This is achieved through a variety of molecules present on the bacterial surface. These molecules, mostly proteins that often form hairlike structures, bind to human tissue at various sites in the body (e.g. throat, lung, guts, skin). An understanding of structure and function of these surface molecules is required for the development of vaccines. Also, if it was possible to prevent the bacteria from binding to the host, it would be possible to intervene with infections at an early stage.
Streptococci are among the most common pathogens affecting humans and animals. Hundreds of millions of cases of strep throat are caused by the bacteria Streptococcus pyogenes every year. So far in countries with good health care penicillin treatment has been effective at keeping this common infection at bay. But as the sudden emergence of resistant pneumococci, C. difficile and MRSA has shown, we need to be prepared for the possibility of S. pyogenes developing penicillin resistance at any time (many strains are already resistant to various other drugs), which could have devastating consequences. For example, if not treated/treatable, strep throat can develop into life-threatening diseases such as rheumatic heart disease (RHD). Despite penicillin availability, RHD is one of the biggest killers of children and youths in India and other developing nations. In RHD our immune system turns against proteins in the body and destroys tissue of the heart valves. This devastating effect is only poorly understood but is known to be linked to a family of proteins (called M proteins) that cover the bacteria in what under the microscope looks like a furry coat.
M proteins were discovered over 80 years ago, and were quickly realised to be required by streptococci for causing disease. Despite their abundance and obvious importance our understanding of how they function is very limited. This lack of progress can be explained with the unusual properties of M proteins, which make them difficult subjects for conventional molecular investigations. We have shown that significant progress can be made using a combination of complementary powerful biophysical techniques (NMR and EPR) that will reveal, at the level of atoms, what M proteins look like and how they work. In this project we will specifically investigate the binding of M proteins to the most abundant human protein, collagen. Collagen is found throughout the body, and its abundance makes it a particularly attractive target for bacteria, and may aid in establishing an infection. Importantly, bacterial binding to collagen may also be the underlying cause of RHD, since collagen is the main component of heart valve tissue. Intriguingly, there are similarities between rheumatic diseases caused by bacterial infections and common autoimmune diseases such as rheumatoid arthritis. The causes of rheumatoid arthritis are unknown but it has been suggested that bacterial infection may play a role. Therefore, our research may uncover interesting links between bacterial infections and other diseases.
A molecular understanding of M proteins can be considered central to the problem of research into streptococcal diseases. Our investigations will ultimately contribute to the development of urgently required new drugs or vaccines for one of the most important and dangerous infectious agents.

Viral and bacterial pathogens have been suggested to trigger autoimmune diseases, but the molecular bases for such long-term consequences of infections are elusive. We propose to study the interaction of the M3 protein from Streptococcus pyogenes with the most abundant human protein, collagen. Binding to collagen is likely to mediate bacterial adhesion, invasion and immune evasion. The M protein:collagen interaction may also form the molecular basis for the induction of anti-collagen antibodies that is observed in the devastating autoimmune disorder rheumatic heart disease. We propose that this autoimmune response is a collagen conformeropathy, caused by exposure of neoepitopes of collagen upon M3 binding.
Due to their architecture (fibrillar triple helical and dimeric coiled coil proteins, respectively) collagens and M proteins have largely been beyond the reach of conventional structural biology. We propose to combine long-range restraints obtainable by PELDOR with short and long-range NMR restraints to map the in-solution conformation of the collagen-binding hypervariable region of the M3 protein. We will use isopep-tagging as a novel strategy to covalently link proteins to generate differentially labelled M3 homodimers for NMR and EPR.
Collagen constructs for M3 binding studies will be designed using Toolkits, libraries of overlapping triple-helical collagen peptides, in particular a new collagen XIII library. The collagen-binding site in M3 will be mapped, and conformational changes in both binding partners will be identified by PELDOR and NMR. Collagen neoepitopes exposed as a result of M3 binding will be identified using scFv antibody phage libraries. These scFvs will be compared to rheumatic heart disease autoantibodies and used in cell-based studies and bacterial adhesion and invasion assays (electron microscopy) aimed at elucidating the biological role of M3-collagen binding for bacterial infections.

Long-term economic and societal impacts of the proposed research are in the improvement of health and well-being, and opportunities for commercialisation and exploitation (both long term and more immediate). Our research will have impact in three distinct areas: 1) fundamental understanding of bacterial virulence factors and host-pathogen interactions; 2) structural biology of challenging proteins; 3) development of therapeutic antibodies and vaccines.

The UK Chief Medical Officer Professor Dame Sally Davies recently warned that the spread of antibiotic resistance and the discovery void in developing novel antimicrobial drugs is an existential threat to the UK and the world. Our basic research into bacterial virulence mechanisms is therefore highly relevant to society with a long-term impact on health. Streptococcus pyogenes (group A streptococci, GAS) is one of the most common and one of the top ten most deadly human pathogens (WHO report 2004). Despite being responsible for over 600 million infections and ~0.5 million deaths annually, streptococcal infections and their sequelae are forgotten among the neglected diseases (PolicyCures, G Finder Report 2010). GAS pathogenicity mechanisms are poorly understood. This hampers urgently needed progress in the development of new antimicrobial agents and vaccines, which is of paramount importance to societies in the UK and elsewhere. We must be prepared for the possibility that an extremely common and potentially devastating pathogen like Streptococcus pyogenes could develop resistance to beta lactams (there is already widespread resistance to other classes of antibiotics), as the experience with pneumococci in the late 20th century showed. Arguably, all attempts to generate a GAS vaccine arguably have failed through a lack of insight into structure and function of the dominant virulence factor, the M protein, which coats the GAS surface. Its molecular architecture makes the M protein a difficult target for structural analysis. The powerful combination of two in-solution structural techniques (NMR and EPR) as proposed here will open new avenues for the molecular analysis of challenging molecular systems that so far have been largely beyond the reach of structural biology.

We will exploit a novel strategy for covalent dimerisation and stabilisation of proteins, the engineering of isopeptide domains that will allow spontaneous and irreversible crosslinking of proteins relying on two short peptide tags. Such a small genetically encoded tool for protein crosslinking will be widely applicable and of great interest to researchers and companies engaged in biotechnology and synthetic biology.

Bacterial adhesion is a recognised target for antimicrobial intervention. A detailed understanding of the underlying molecular mechanisms is urgently required to facilitate the development of new antimicrobial agents and vaccines, which is of paramount importance to society in the UK and elsewhere. We believe that collagen, the most abundant vertebrate protein, may be an important substrate for S. pyogenes adhesion. This project will identify in molecular detail specific sites in the collagens to which streptococci can bind through the M protein, thus offering the M3:collagen peptide interaction as a druggable target, readily adaptable to high-throughput screening in a commercial setting. Perhaps most significantly, our research will yield single-chain antibodies that have therapeutic potential for the treatment of post-streptococcal sequelae. Antibody therapies have been successfully employed for invasive streptococcal diseases such as necrotising fasciitis and toxic shock, where no other treatment options are available.