Methylome-dependent transformation of emm1 group A streptococci.

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A Streptococcus (GAS), a lethal human pathogen currently associated with an unprecedented surge of infections worldwide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this "restriction barrier" thus presents a major technical challenge. Here, we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Furthermore, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associated emm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol, whereby the restriction barrier is overcome by the addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAG strains including clinical isolates representing all emm1 lineages and will expedite critical research interrogating the genetics of emm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation and the importance of defining lineage-specific mechanisms of genetic recalcitrance. IMPORTANCE Understanding the mechanisms by which bacterial pathogens are able to cause disease is essential to enable the targeted development of novel therapeutics. A key experimental approach to facilitate this research is the generation of bacterial mutants, through either specific gene deletions or sequence manipulation. This process relies on the ability to transform bacteria with exogenous DNA designed to generate the desired sequence changes. Bacteria have naturally developed protective mechanisms to detect and destroy invading DNA, and these systems severely impede the genetic manipulation of many important pathogens, including the lethal human pathogen group A Streptococcus (GAS). Many GAS lineages exist, of which emm1 is dominant among clinical isolates. Based on new experimental evidence, we identify the mechanism by which transformation is impaired in the emm1 lineage and establish an improved and highly efficient transformation protocol to expedite the generation of mutants.

Structure of the Streptococcus pyogenes NAD+ Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes.

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a ß-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

Extracellular bacterial lymphatic metastasis drives Streptococcus pyogenes systemic infection.

Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity.

Flow Cytometry-Based Assays to Quantify Complement Deposition and Neutrophil Uptake of Group A Streptococcus.

Quantification of complement deposition and subsequent neutrophil-mediated uptake provides an important way to assess the role of different bacterial factors in evasion of the host innate immune response. Here, we describe flow cytometry-based methods to allow quantification of deposition of the complement opsonin C3 on the bacterial surface and subsequent uptake by primary human neutrophils. The assays outlined below provide key methods to determine whether specific bacterial factors are involved in the evasion of complement-mediated immunity, using widely accessible reagents and equipment.

Emergence of dominant toxigenic M1T1 Streptococcus pyogenes clone during increased scarlet fever activity in England: a population-based molecular epidemiological study.

BACKGROUND: Since 2014, England has seen increased scarlet fever activity unprecedented in modern times. In 2016, England's scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular epidemiological investigation of these events. METHODS: We analysed changes in S pyogenes emm genotypes, and notifications of scarlet fever and invasive disease in 2014-16 using regional (northwest London) and national (England and Wales) data. Genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009-16 were analysed and compared with 2800 global emm1 sequences. Transcript and protein expression of streptococcal pyrogenic exotoxin A (SpeA; also known as scarlet fever or erythrogenic toxin A) in sequenced, non-invasive emm1 isolates was quantified by real-time PCR and western blot analyses. FINDINGS: Coincident with national increases in scarlet fever and invasive disease notifications, emm1 S pyogenes upper respiratory tract isolates increased significantly in northwest London in the March to May period, from five (5%) of 96 isolates in 2014, to 28 (19%) of 147 isolates in 2015 (p=0·0021 vs 2014 values), to 47 (33%) of 144 in 2016 (p=0·0080 vs 2015 values). Similarly, invasive emm1 isolates collected nationally in the same period increased from 183 (31%) of 587 in 2015 to 267 (42%) of 637 in 2016 (p<0·0001). Sequences of emm1 isolates from 2009-16 showed emergence of a new emm1 lineage (designated M1UK)-with overlap of pharyngitis, scarlet fever, and invasive M1UK strains-which could be genotypically distinguished from pandemic emm1 isolates (M1global) by 27 single-nucleotide polymorphisms. Median SpeA protein concentration in supernatant was nine-times higher among M1UK isolates (190·2 ng/mL [IQR 168·9-200·4]; n=10) than M1global isolates (20·9 ng/mL [0·0-27·3]; n=10; p<0·0001). M1UK expanded nationally to represent 252 (84%) of all 299 emm1 genomes in 2016. Phylogenetic analysis of published datasets identified single M1UK isolates in Denmark and the USA. INTERPRETATION: A dominant new emm1 S pyogenes lineage characterised by increased SpeA production has emerged during increased S pyogenes activity in England. The expanded reservoir of M1UK and recognised invasive potential of emm1 S pyogenes provide plausible explanation for the increased incidence of invasive disease, and rationale for global surveillance. FUNDING: UK Medical Research Council, UK National Institute for Health Research, Wellcome Trust, Rosetrees Trust, Stoneygate Trust.

RocA Binds CsrS To Modulate CsrRS-Mediated Gene Regulation in Group A Streptococcus.

The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation.IMPORTANCE Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.

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.

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

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 commonly expressed exoproteins and proteolytic cleavage events by proteomic mining of clinically relevant UK isolates of Staphylococcus aureus.

The range of exoproteins and core exoproteome of 14 Staphylococcus aureus isolates representing major lineages associated with asymptomatic carriage and clinical disease in the UK was identified by MS proteomics using a combined database incorporating sequences derived from 39 S. aureus genomes. In all, 632 different proteins were identified and, of these, only 52 (8 %) were found in all 14 isolates whereas 144 (23 %) were found in just a single isolate. Comparison of the observed mass of each protein (based on migration by SDS-PAGE) with its predicted mass (based on amino acid sequence) suggested that 95 % of the proteins identified were not subject to any major post-translational modification. Migration of 5 % of the proteins was not as expected: 1 % of the proteins migrated at a mass greater than predicted, while 4 % appeared to have undergone proteolytic cleavage; these included SsaA2, Aur, SspP, Ebh as well as BlaR1, MecR1, FsH, OatA and LtaS. Intriguingly, a truncated SasG was produced by a single CC8 USA300-like strain. The analysis provided evidence of the marked heterogeneity in protein expression by S. aureus in broth, while yielding a core but narrow common exoproteome.

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.

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.

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.

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.