Generation of tools for expression and purification of the phage-encoded Type I restriction enzyme inhibitor, Ocr.

DNA manipulation is an essential tool in molecular microbiology research that is dependent on the ability of bacteria to take up and preserve foreign DNA by horizontal gene transfer. This process can be significantly impaired by the activity of bacterial restriction modification systems; bacterial operons comprising paired enzymatic activities that protectively methylate host DNA, while cleaving incoming unmodified foreign DNA. Ocr is a phage-encoded protein that inhibits Type I restriction modification systems, the addition of which significantly improves bacterial transformation efficiency.

Methylome-dependent transformation of 1 group A streptococci.

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A (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.

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.

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.

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.

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

The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A 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 and 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.

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.

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

The range of exoproteins and core exoproteome of 14 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 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.

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.

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.

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.

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.

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.

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.