Nicola N. Lynskey

New understandings in Streptococcus pyogenes.

Purpose of review A resurgence of invasive group A streptococcal infections highlights the need for better knowledge of streptococcal biology. This review summarizes the recent advances in our understanding of the field. Recent findings Invasive group A streptococcal infections cause significant morbidity and mortality worldwide. The current upsurge of invasive infections in developed countries is predominantly linked to the spread of a clonal hypervirulent population of M1T1 serotype strains (emm1), although sporadic increases in other types have been reported, including emm3 strains in the UK, and emm28 strains among cases of puerperal sepsis. Mutations of a regulatory system, CovR/S (control of virulence), are important in the transition of emm1 strains from noninvasive to invasive phenotype. New research has been undertaken to identify major virulence factors that typify the invasive phenotype. In less-developed regions, the importance of rheumatic carditis and need for a vaccine that addresses a much wider range of streptococcal emm types predominates research efforts. Summary Advances in molecular technology have furthered our understanding of virulence factors that underpin group A streptococcus invasiveness. The increased prevalence of invasive disease coupled with the devastating effects of chronic rheumatic heart disease, affecting predominantly low-income regions, underline the need for the development of an effective vaccine.

RocA Truncation Underpins Hyper-Encapsulation, Carriage Longevity and Transmissibility of Serotype M18 Group A Streptococci

Group A streptococcal isolates of serotype M18 are historically associated with epidemic waves of pharyngitis and the non-suppurative immune sequela rheumatic fever. The serotype is defined by a unique, highly encapsulated phenotype, yet the molecular basis for this unusual colony morphology is unknown. Here we identify a truncation in the regulatory protein RocA, unique to and conserved within our serotype M18 GAS collection, and demonstrate that it underlies the characteristic M18 capsule phenotype. Reciprocal allelic exchange mutagenesis of rocA between M18 GAS and M89 GAS demonstrated that truncation of RocA was both necessary and sufficient for hyper-encapsulation via up-regulation of both precursors required for hyaluronic acid synthesis. Although RocA was shown to positively enhance covR transcription, quantitative proteomics revealed RocA to be a metabolic regulator with activity beyond the CovR/S regulon. M18 GAS demonstrated a uniquely protuberant chain formation following culture on agar that was dependent on excess capsule and the RocA mutation. Correction of the M18 rocA mutation reduced GAS survival in human blood, and in vivo naso-pharyngeal carriage longevity in a murine model, with an associated drop in bacterial airborne transmission during infection. In summary, a naturally occurring truncation in a regulator explains the encapsulation phenotype, carriage longevity and transmissibility of M18 GAS, highlighting the close interrelation of metabolism, capsule and virulence.

Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction.

The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology.

A Truncation in the Regulator RocA Underlies Heightened Capsule Expression in Serotype M3 Group A Streptococci

The recent report from Cao et al. regarding hyper-virulence associated with M3 group A streptococcal (GAS) infections identified serotype-specific mutations in two important gene regulators, fasC and rivR . These mutations were demonstrated to underlie the differential expression of two important

Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG

Summary Objectives Despite over a century of research and the careful scrutiny of many promising targets, there is currently no vaccine available for the prevention of Streptococcus pyogenes infection. Through analysis of the protective, anti-streptococcal components of pooled human immunoglobulin, we previously identified ten highly conserved and invariant S. pyogenes antigens that contribute to anti-streptococcal immunity in the adult population. We sought to emulate population immunity to S. pyogenes through a process of active vaccination, using the antigens targeted by pooled human immunoglobulin. Methods Seven targets were produced recombinantly and mixed to form a multicomponent vaccine (Spy7). Vaccinated mice were challenged with S. pyogenes isolates representing four globally relevant serotypes (M1, M3, M12 and M89) using an established model of invasive disease. Results Vaccination with Spy7 stimulated the production of anti-streptococcal antibodies, and limited systemic dissemination of M1 and M3 S. pyogenes from an intramuscular infection focus. Vaccination additionally attenuated disease severity due to M1 S. pyogenes as evidenced by reduction in weight loss, and modulated cytokine release. Conclusion Spy7 vaccination successfully stimulated the generation of protective anti-streptococcal immunity in vivo. Identification of reactive antigens using pooled human immunoglobulin may represent a novel route to vaccine discovery for extracellular bacteria.

Identification of the Streptococcus pyogenes surface antigens recognised by pooled human immunoglobulin.

Immunity to common bacteria requires the generation of antibodies that promote opsonophagocytosis and neutralise toxins. Pooled human immunoglobulin is widely advocated as an adjunctive treatment for clinical Streptococcus pyogenes infection however, the protein targets of the reagent remain ill defined. Affinity purification of the anti-streptococcal antibodies present within pooled immunoglobulin resulted in the generation of an IgG preparation that promoted opsonophagocytic killing of S. pyogenes in vitro and provided passive immunity in vivo. Isolation of the streptococcal surface proteins recognised by pooled human immunoglobulin permitted identification and ranking of 94 protein antigens, ten of which were reproducibly identified across four contemporary invasive S. pyogenes serotypes (M1, M3, M12 and M89). The data provide novel insight into the action of pooled human immunoglobulin during invasive S. pyogenes infection, and demonstrate a potential route to enhance the efficacy of antibody based therapies.

Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis.

Modification of the classical Lancefield assay of group A streptococcal killing to reduce inter-donor variation

The lack of a surrogate-of-immunity assay presents a major barrier to Streptococcus pyogenes research. Modification of the Lancefield assay to include an antibody digestion step reduced inter-donor variation and permitted detection of the anti-streptococcal activity of intravenous immunoglobulin and convalescent serum, thus facilitating retrospective evaluation of immunity using stored samples.

The effect of trauma on invasive group A streptococcal disease

BACKGROUND Necrotising fasciitis due to invasive group A streptococcus (iGAS) is frequently associated with type emm1 isolates, with an attendant mortality of 40%. Some cases occur in previously healthy individuals with a history of upper respiratory tract infection, soft tissue contusion, and no obvious portal of entry. Using a new model of mild contusion injury, we set out to determine the effect of contusion on iGAS bacterial burden, phenotype, and host cytokine response. METHODS A new model of mild contusion was developed using a weight drop device and characterised in two strains of mice, CD1 and FVB/n. The effect of contusion on emm1 iGAS infection was assessed in three murine models of infection: lower respiratory tract (intranasal challenge of 1 × 10(7) colony forming units [CFU] per mouse), intravenous (1 × 10(7)· per mouse via the lateral tail vein), and muscle (1 × 10(8) CFU per mouse intramuscularly) at three timepoints after injury (24, 48, and 72 h). Bacterial burden, host cytokine response, and histological changes were analysed. Further molecular work was performed to assess the change in bacterial morphology observed after contusion injury in the muscle model. Mann-Whitney U test was used to compare differences in bacterial burden and cytokine responses between trauma and control groups. FINDINGS Application of a force of 15·7 mJ resulted in histological changes in muscle consistent with mild trauma with no evidence of overlying skin injury, no bony injury, and minimum cytokine response. Contusion to soft tissue had no effect on bacterial burden or cytokine response in a mouse model of systemic infection (after intravenous inoculation) at three timepoints. Despite bacteraemia, specific seeding of the contused tissue did not occur in this model. By contrast, blunt contusion affected progression of a subsequent local GAS muscle infection and increased dissemination to blood in the lower respiratory tract infection model. Specifically, contusion increased emm1 GAS dissemination locally to draining lymph nodes (controls median 183 CFU per node [IQR 8-5800] vs trauma group 20 000 [1875-601 250]). Dissemination to lymph node was linked to a phenotypic change in bacterial capsule morphology. This phenotypic change was stable despite passage, consistent with a genetic change, and was associated with an increase in bacterial hyaluronan production (mucoid colonies 200 μg per CFU and no detectable capsule production in the non-mucoid colonies). INTERPRETATION We found that non-penetrating trauma was associated with an enhanced susceptibility to invasive GAS disease. This model of mild contusion did not provide a focus for initiation or seeding of bacteraemic infection but instead provided an environment that determined the phenotype of the bacteria and enhanced local dissemination after iGAS infection at the same site. The environmental and genetic cues underlying dissemination are the subject of continuing research. FUNDING Royal Army Medical Corps, Surgeon Generals Research Strategy Group, Ministry of Defence.

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death, and reduced immunoglobulin release

Background Streptococcal pyrogenic exotoxin (SPE)A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leukocyte profile of tonsil differs from peripheral blood, the impact of SPEA production on human tonsil cell function was investigated. Methods Human tonsil cells from routine tonsillectomy, were co-incubated with purified streptococcal superantigens or isogenic streptococcal culture supernatants, differing only in superantigen content. Tonsil cell proliferation was quantified by tritium-incorporation, and cell surface characteristics assessed by flow-cytometry. Soluble mediators were measured using ELISA and quantitative (q)RT-PCR was performed for immunoglobulin gene expression. Results Tonsil T cells proliferated in response to SPEA and demonstrated typical release of pro-inflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7d. In contrast, marked B cell apoptosis and abrogation of total IgA, IgM, and IgG production occurred in the presence of SPEA and other superantigens. In SPEA-stimulated cultures, T follicular helper (TfH) cells showed a reduction in CXCR5 expression, but up-regulation of CD134 (OX40), CD278 (ICOS) and CD150 (SLAM) expression, indicative of a phenotypic change in the TfH population and associated with impaired chemotactic response to CXCL13. Conclusions SPEA and other superantigens cause dysregulated tonsil immune function, driving T cells from TFH to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a likely survival advantage.