Mapping Autoantibodies in Children With Acute Rheumatic Fever.

Background: Acute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease. Methods: This study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand. Results: Global analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p < 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens. Conclusions: The broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

Antibody responses to collagen peptides and streptococcal collagen-like 1 proteins in acute rheumatic fever patients.

Acute rheumatic fever (ARF) is a serious post-infectious immune sequelae of Group A streptococcus (GAS). Pathogenesis remains poorly understood, including the events associated with collagen autoantibody generation. GAS express streptococcal collagen-like proteins (Scl) that contain a collagenous domain resembling human collagen. Here, the relationship between antibody reactivity to GAS Scl proteins and human collagen in ARF was investigated. Serum IgG specific for a representative Scl protein (Scl1.1) together with collagen-I and collagen-IV mimetic peptides were quantified in ARF patients (n = 36) and healthy matched controls (n = 36). Reactivity to Scl1.1 was significantly elevated in ARF compared to controls (P < 0.0001) and this was mapped to the collagen-like region of the protein, rather than the N-terminal non-collagenous region. Reactivity to collagen-1 and collagen-IV peptides was also significantly elevated in ARF cases (P < 0.001). However, there was no correlation between Scl1.1 and collagen peptide antibody binding, and hierarchical clustering of ARF cases by IgG reactivity showed two distinct clusters, with Scl1.1 antigens in one and collagen peptides in the other, demonstrating that collagen autoantibodies are not immunologically related to those targeting Scl1.1. Thus, anti-collagen antibodies in ARF appear to be generated as part of the autoreactivity process, independent of any mimicry with GAS collagen-like proteins.

Development and Evaluation of a New Triplex Immunoassay That Detects Group A Streptococcus Antibodies for the Diagnosis of Rheumatic Fever.

Streptococcal serology is a cornerstone in the diagnosis of acute rheumatic fever (ARF), a postinfectious sequela associated with group A Streptococcus infection. Current tests that measure anti-streptolysin O (ASO) and anti-DNaseB (ADB) titers require parallel processing, with their predictive value limited by the low rate of decay in antibody response. Accordingly, our objective was to develop and assess the diagnostic potential of a triplex bead-based assay, which simultaneously quantifies ASO and ADB together with titers for a third antigen, SpnA. Our previous cytometric bead assay was transferred to the clinically appropriate Luminex platform by coupling streptolysin O, DNaseB, and SpnA to spectrally unique magnetic beads. Sera from more than 350 subjects, including 97 ARF patients, were used to validate the assay and explore immunokinetics. Operating parameters demonstrate that the triplex assay produces accurate and reproducible antibody titers which, for ASO and ADB, are highly correlative with existing assay methodology. When ARF patients were stratified by time (days following hospital admission), there was no difference in ASO and ADB between <28 and 28+ day groups. However, for anti-SpnA, there was a significant decrease (P < 0.05) in the 28+ day group, indicative of faster anti-SpnA antibody decay. Anti-SpnA immunokinetics support very recent group A Streptococcus infection and may assist in diagnostic classification of ARF. Further, bead-based assays enable streptococcal serology to be performed efficiently in a high-throughput manner.

Systems immunology reveals a linked IgG3-C4 response in patients with acute rheumatic fever.

Acute rheumatic fever (ARF) and chronic rheumatic heart disease (RHD) are autoimmune sequelae of a Group A streptococcal infection with significant global mortality and poorly understood pathogenesis. Immunoglobulin and complement deposition were observed in ARF/RHD valve tissue over 50 years ago, yet contemporary investigations have been lacking. This study applied systems immunology to investigate the relationships between the complement system and immunoglobulin in ARF. Patients were stratified by C-reactive protein (CRP) concentration into high (≥10 µg mL-1 ) and low (<10 µg mL-1 ) groups to distinguish those with clinically significant inflammatory processes from those with abating inflammation. The circulating concentrations of 17 complement factors and six immunoglobulin isotypes and subclasses were measured in ARF patients and highly matched healthy controls using multiplex bead-based immunoassays. An integrative statistical approach combining feature selection and principal component analysis revealed a linked IgG3-C4 response in ARF patients with high CRP that was absent in controls. Strikingly, both IgG3 and C4 were elevated above clinical reference ranges, suggesting these features are a marker of ARF-associated inflammation. Humoral immunity in response to M protein, an antigen implicated in ARF pathogenesis, was completely polarized to IgG3 in the patient group. Furthermore, the anti-M-protein IgG3 response was correlated with circulating IgG3 concentration, highlighting a potential role for this potent immunoglobulin subclass in disease. In conclusion, a linked IgG3-C4 response appears important in the initial, inflammatory stage of ARF and may have immediate utility as a clinical biomarker given the lack of specific diagnostic tests currently available.

The uncharacterized bacterial protein YejG has the same architecture as domain III of elongation factor G.

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.

The novel Group A Streptococcus antigen SpnA combined with bead-based immunoassay technology improves streptococcal serology for the diagnosis of acute rheumatic fever.

OBJECTIVES: Streptococcal serology provides evidence of prior Group A Streptococcus (GAS) exposure, crucial to the diagnosis of acute rheumatic fever (ARF) and post-streptococcal glomerulonephritis. However, current tests, which measure anti-streptolysin-O and anti-DNaseB antibodies, are limited by false positives in GAS endemic settings, and incompatible methodology requiring the two tests to be run in parallel. The objective was to improve streptococcal serology by combining the novel GAS antigen, SpnA, with streptolysin-O and DNaseB in a contemporary, bead-based immunoassay. METHODS: Recombinant streptolysin-O, DNAseB and SpnA were conjugated to polystyrene beads with unique fluorescence positions so antibody binding to all three antigens could be detected simultaneously by cytometric bead array. Multiplex assays were run on sera collected in three groups: ARF; ethnically matched healthy children; and healthy adults. RESULTS: The ability of the antigens to detect a previous GAS exposure in ARF was assessed using the 80th centile of the healthy children group as cut-off (upper limit of normal). SpnA had the highest sensitivity at 88%, compared with 75% for streptolysin-O and 56% for DNaseB. CONCLUSIONS: SpnA has favorable immunokinetics for streptococcal serology, and can be combined with anti-streptolysin-O and anti-DNaseB in a multiplex format to improve efficiency and accuracy.

Error-prone PCR and effective generation of gene variant libraries for directed evolution.

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.

Construction and analysis of randomized protein-encoding libraries using error-prone PCR.

In contrast to site-directed mutagenesis and rational design, directed evolution harnesses Darwinian principles to identify proteins with new or improved properties. The critical first steps in a directed evolution experiment are as follows: (a) to introduce random diversity into the gene of interest and (b) to capture that diversity by cloning the resulting population of molecules into a suitable expression vector, en bloc. Error-prone PCR (epPCR) is a common method for introducing random mutations into a gene. In this chapter, we describe detailed protocols for epPCR and for the construction of large, maximally diverse libraries of cloned variants. We also describe the utility of an online program, PEDEL-AA, for analyzing the compositions of epPCR libraries. The methods described here were used to construct several libraries in our laboratory. A side-by-side comparison of the results is used to show that, ultimately, epPCR is a highly stochastic process.

Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli.

Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (≤16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004-17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations.