Group A streptococcus (GAS) is responsible for a wide range of diseases ranging from superficial infections, such as pharyngitis and impetigo, to life-threatening diseases, such as toxic shock syndrome and acute rheumatic fever (ARF). GAS pili are hair-like extensions protruding from the cell surface and consist of highly immunogenic structural proteins: the backbone pilin (BP) and one or two accessory pilins (AP1 and AP2). The protease-resistant BP builds the pilus shaft and has been recognized as the T-antigen, which forms the basis of a major serological typing scheme that is often used as a supplement to M typing. A previous sequence analysis of the bp gene (tee gene) in 39 GAS isolates revealed 15 different bp/tee types. In this study, we sequenced the bp/tee gene from 100 GAS isolates obtained from patients with pharyngitis, ARF or invasive disease in New Zealand. We found 20 new bp/tee alleles and four new bp/tee types/ subtypes. No association between bp/tee type and clinical outcome was observed. We confirmed earlier reports that the emm type and tee type are associated strongly, but we also found exceptions, where multiple tee types could be found in certain M/emm type strains, such as M/ emm89. We also reported, for the first time, the existence of a chimeric bp/tee allele, which was assigned into a new subclade (bp/tee3.1). A strong sequence conservation of the bp/tee gene was observed within the individual bp/tee types/subtypes (.97 % sequence identity), as well as between historical and contemporary New Zealand and international GAS strains. This temporal and geographical sequence stability provided further evidence for the potential use of the BP/Tantigen as a vaccine target.
We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.T wo of the most significant consequences of group A streptococcal (GAS) infection are acute rheumatic fever (ARF) and its sequelae, rheumatic heart disease (RHD). New Zealand has among the highest incidences of ARF in the developed world, with the greatest burden of disease in indigenous New Zealand (Ma ori) and Pacific populations (1).Contemporary molecular typing of GAS is carried out by sequence analysis of the hypervariable region of the emm gene that encodes the M protein (2). Studies in the United States have suggested an association between distinct GAS emm types (so-called rheumatogenic strains, such as emm3, emm5, emm6, and emm18) and ARF (3). However, more recent epidemiological studies in areas where ARF is common today have found that ARF is not restricted to these rheumatogenic strains (4), raising questions around the concept of rheumatogenicity and emm type. It has further been postulated that certain GAS emm types (most notably emm3) may be rheumatogenic due to the presence of a specific collagen-binding motif, designated peptide associated with rheumatic fever (PARF), which elicits an immune response to type IV collagen (5, 6). To date, however, the presence of the PARF motif has not been systematically assessed in a large collection of GAS strains temporally associated with ARF.Recently, an Australian and New Zealand GAS vaccine development program (the Coalition to Accelerate New Vaccines Against Streptococcus [CANVAS]) was formed with the aim of identifying suitable vaccine GAS candidates for both the Australian and New Zealand settings, and more widely (7). At present, the most clinically advanced GAS vaccine candidates are those that target the N-terminal region of the M protein, such as an experimental 30-valent M-protein vaccine (8). While this vaccine includes the classical rheumatogenic emm types, there have been few analyses to inform the coverage of contemporary ARF strains. Recently, an emm cluster-based typing system that classifies known emm types into 48 related emm clusters has been applied to several collections of GAS isolates and has shed new insights into the epidemiology of GAS and the potential vaccine coverage of M-protein-based vaccines (9, 10). The emm cluster system also predicts an emm pattern type that in turn correlates well with tissue tropism (pattern A-C for pharyngeal, pattern D for skin, and pattern E for either) (9). Accordingly, the aims of this study were to (i) compare the molecular epidemiology and theoretical vaccine coverage of GAS isolates associated with ARF in New Zealand with those of GAS isolates recovered from other GAS-related clinical syndromes, and (ii) identify GAS isolates containing the PARF motif and associate the pre...
Acute rheumatic fever (ARF) is an autoimmune response to Group A Streptococcus (GAS) infection. Repeated GAS exposures are proposed to ‘prime’ the immune system for autoimmunity. This notion of immune-priming by multiple GAS infections was first postulated in the 1960s, but direct experimental evidence to support the hypothesis has been lacking. Here, we present novel methodology, based on antibody responses to GAS T-antigens, that enables previous GAS exposures to be mapped in patient sera. T-antigens are surface expressed, type specific antigens and GAS strains fall into 18 major clades or T-types. A panel of recombinant T-antigens was generated and immunoassays were performed in parallel with serum depletion experiments allowing type-specific T-antigen antibodies to be distinguished from cross-reactive antibodies. At least two distinct GAS exposures were detected in each of the ARF sera tested. Furthermore, no two sera had the same T-antigen reactivity profile suggesting that each patient was exposed to a unique series of GAS T-types prior to developing ARF. The methods have provided much-needed experimental evidence to substantiate the immune-priming hypothesis, and will facilitate further serological profiling studies that explore the multifaceted interactions between GAS and the host.
The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.
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